Method for detecting and reversing resistance to macrocyclic lactone compounds

ABSTRACT

This invention describes novel purified and isolated nucleic acid molecules or the fragments thereof, extracted from nematode or arthropod pests or recombinant, which encode P-glycoprotein homologs and regulate resistance to the macrocyclic lactone compounds. The invention further relates to the new P-glycoprotein homolog expression product of these nucleic acids. Also described herein are methods for detecting the gene encoding for resistance to the macrocyclic lactone compounds in nematode or arthropod pests which comprise comparing the nucleic acids extracted from a pest specimen to the nucleic acids encoding for resistance and the nucleic acids encoding for susceptibility to the macrocyclic lactone compounds. Lastly, the present invention is also drawn to methods and compositions for increasing the efficacy of the macrocyclic lactone compounds against resistant nematode or resistant arthropod pests which comprise administering to a mammal or applying to crops and the like a pesticidal enhancing effective amount of a multidrug resistance reversing agent.

BACKGROUND OF THE INVENTION RELATED U.S. APPLICATION DATA

[0001] This application claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application No. 60/045,160, filed Apr. 30, 1997.

FIELD OF THE INVENTION

[0002] This invention relates generally to novel methods for diagnosingand overcoming resistance to the macrocyclic lactone compounds. Morespecifically, the invention pertains to unique methods for detecting thedevelopment of resistance to macrocyclic lactones using nucleic acidprobes and enhancing the efficacy of the macrocyclic lactones usingmultidrug resistant reversing agents.

DESCRIPTION OF RELATED ART

[0003] Macrocyclic lactone compounds such as the LL-F28249 compounds,the milbemycins and the avermectins are widely used for treatment ofnematode and arthropod parasites. The highly active LL-F28249 family ofcompounds are natural endectocidal agents isolated from the fermentationbroth of Streptomyces cyaneogriseus subsp. noncyanogenus. U.S. Pat. No.5,106,994 and its continuation U.S. Pat. No. 5,169,956 describe thepreparation of the major and minor components, LL-F28249α-λ. TheLL-F28249 family of compounds further includes, but is not limited to,the semisynthetic 23-oxo derivatives and 23-imino derivatives ofLL-F28249α-λ which are shown in U.S. Pat. No. 4,916,154. Moxidectin,chemically known as 23-(O-methyloxime)-LL-F28249α, is a particularlypotent 23-imino derivative. Other examples of LL-F28249 derivativesinclude, but are not limited to,23-(O-methyloxime)-5-(phenoxyacetoxy)-LL-F28249α,23-(semicarbazone)-LL-F28249α and 23-(thiosemicarbazone)-LL-F28249α.

[0004] The milbemycins, also known as the B-41 series of antibiotics,are naturally occurring macrocyclic lactones isolated from themicroorganism, Streptomyces hygroscopicus subsp. aureolacrimosus. U.S.Pat. No. 3,950,360 shows the preparation of the macrolide antibioticsmilbemycin_(α1-α10), milbemycin_(β1-β3) etc. These compounds are alsocommonly referred to as milbemycin A, milbemycin B, milbemycin D and thelike, or antibiotic B-41A1, antibiotic B-41A3, etc.

[0005] The avermectins, also known as the C-076 family of compounds, arenaturally occurring macrocyclic lactones produced by the soilactinomycete microorganism, Streptomyces avermitilis. U.S. Pat. No.4,310,519 discloses the isolation and preparation of the majorcomponents A_(1a) (e.g., avermectin A_(1a)), A_(2a), B_(1a) and B_(2a),and the minor components A_(1b) (e.g., avermectin A_(1b)), A_(2b),B_(1b) and B_(2b). The C-076 family additionally embraces thesemisynthetic derivatives such as the 22,23-dihydroavermectins describedin U.S. Pat. No. 4,199,569. The semisynthetic derivatives include, butare not limited to, ivermectin, abamectin, doramectin, eprinomectin andthe like.

[0006] Resistance to all of the broad spectrum macrocyclic lactonecompounds has been encountered in most regions of the world where thecompounds are used routinely in animal production. For instance, drugresistance to ivermectin (IVM), chemically known as22,23-dihydroavermectin B₁ or 22,23-dihydro C-076 B₁ and a commonly usedmember of the avermectin drug family, has become a widespread problem,particularly in nematodes of sheep, goats and cattle (Shoop, Parasitol.Today 9: 154-159, 1993). In some parts of the world, the survival ofcommercial animal production is threatened by the development ofanthelmintic resistance. Additionally, there is conflicting evidence asto whether ivermectin (avermectin) resistance confers resistance to therelated milbemycins or other macrolides (Arena et al., J. Parasitol. 81:286-294, 1995; Oosthuizen and Erasmus, J. So. African Vet. Assoc. 64:9-12, 1993; Pomroy and Whelan, Vet. Rec. 132: 416, 1993; Shoop, 1993;Condora et al., Vet. Rec. 132: 651-652, 1993; Pomroy et al., N.Z. Vet.J. 40: 76, 1992; Pankavich et al., Vet. Rec. 130: 241-242, 1992; Craiget al., Vet. Parasitol. 41: 329-333, 1992). The mechanisms of resistanceto the avermectins, the milbemycins and other macrocyclic lactonecompounds remain unknown.

[0007] P-glycoproteins (Pgp) were identified some years ago as proteinsinvolved in multidrug resistance (MDR) of mammalian tumor cells (Julinoand Ling, 1976; Gros and Buschman, 1993; Gotteesman and Pastan, 1993).MDR proteins may also be involved in drug resistance in the protozoalparasites Entamoeba histolytica (Whirth, Archivos De InvestigacionMedica 21(Supp. 1): 183-189, 1990; Samuelson et al., Mol. Biochem.Parasitol. 38: 281-290, 1990), Leishmania enriietti (Chow, Mol. Biochem.Parasitol. 60: 195-208, 1993), L. dononani (Callahan et al., Mol.Biochem. Parasitol. 68: 145-149, 1994); and Plasmodium falciparum(Volkman et al., Mol. Biochem. Parasitol. 57: 203-211, 1993; Cowman etal., J. Cell Biol. 113: 1033-1042, 1991). While many researchers believethat the proposed mechanism for Pgp involvement in drug resistance isthat Pgp behaves as a pump to increase drug efflux, Callahan et al.(1994) suggested that Pgp may work by decreasing drug influx. However,the whole picture of how Pgp can be responsible for drug resistance isstill unclear.

[0008] Only recently have Pgp homologs been investigated in nematodes(Sangster, Parasitol. Today 10: 319-322, 1994; Lincke et al., EMBO J.12: 1615-1620, 1993; Lincke et al., J. Mol. Biol. 228: 701-711, 1992).Three full length Pgp genes and one partial Pgp gene from thefree-living nematode, Caenorhabditis elegans have been cloned, sequencedand mapped to chromosomes I, IV and X (Lincke et al., 1992). Sangster etal., J. Cell Biochem. 17 (Supp.): 1223, 1993, indicated evidence forseveral partial genes for Pgp in the parasitic nematode Haemonchuscontortus, although sequence information was missing. In vivoexperiments have shown that disruption of the mouse mdr1, aP-glycoprotein gene, leads to an impairment in the blood-brain barrierand to increased sensitivity to drugs in these mice (Schinkel et al.,Cell 77:491-502, 1994). Mice with deletion of mcdr1a were 50-100 timesmore sensitive to ivermectin than normal mice.

[0009] Drug resistance based on overexpression of P-glycoprotein hasbeen shown to be reversed by verapamil and a number of other calciumchannel blockers, calmodulin antagonists, steroids and hormonal analogs,cyclosporins, dipyridamole and other MDR-reversing agents (Ford,Hematol. Oncol. Clin. North Am. 9: 337-361, 1995). However, there hasbeen no report or suggestion in the literature to use MDR-reversingagents to combat resistance in nematodes and arthropods to pesticides.

[0010] There is a definite need to understand the mechanism ofmacrocyclic lactone resistance, to be able to detect insipientresistance before it becomes flagrant and is difficult to manage thehealth of the animals. The ability to reverse the resistance has greatpotential for maintaining parasite control in the face of a failure ofconventional treatment. An important object of the present invention,thus, is to determine these mechanisms of resistance in order to findviable, sensitive means to detect and to overcome the problematicresistance thereby improving parasite control.

BRIEF SUMMARY OF THE INVENTION

[0011] Heretofore unknown, it is now found that the mechanism ofresistance to the macrocyclic lactone compounds is due to overexpressionof novel P-glycoprotein homologs. It is further newly found that thenucleic acid molecules encoding the P-glycoprotein homologs or thefragments thereof regulating this resistance are useful as unique probesin methods for diagnosing the resistance to the macrocyclic lactones.For the first time, the reversal of resistance to the macrocycliclactone compounds using multidrug resistance reversing agents isdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The background of the invention and its departure from the artwill be further described hereinbelow with reference to the accompanyingdrawings, wherein:

[0013]FIG. 1 shows the 432 bp PCR product which is generated from aHaemonchus contortus cDNA PBLUESCRIPT® library as template anddegenerate primers based on the conserved ATP binding domains ofCaenorhabditis elegans P-glycoprotein genes after electrophoresis on anagarose gel.

[0014]FIGS. 2A and 2B represent, respectively, the nucleotide sequenceof the 432 bp PCR product shown in FIG. 1 and the predicted amino acidtranslation of the cDNA (which correspond to SEQ ID NO:1 and SEQ IDNO:2, respectively).

[0015]FIG. 3 shows the autoradiographs of the Northern blots of RNAextracted from eggs of ivermectin sensitive and resistant (MKIS andMKIR; ACIS and ACIR) nematode strains respectively. The [³²P]-432 bp PCRproduct, with homology to Pgp, is used as one probe and a [³²P]-actinfragment from pBAl is used as a second probe.

[0016]FIGS. 4A to 4B represent the full-length cDNA sequence (4175 bp)of the PGP-A clone from the H. contortus cDNA library with high homologyto known P-glycoproteins (which corresponds to SEQ ID NO:3).

[0017]FIG. 5 represents the partial cDNA sequence (1810 bp) of the 5′end of the PGP-A clone from the H. contortus cDNA library (whichcorresponds to SEQ ID NO:4).

[0018]FIG. 6 represents the partial cDNA sequence (2698 bp) of the 3′end of the PGP-A clone from the H. contortus cDNA library (whichcorresponds to SEQ ID NO:5).

[0019]FIG. 7 represents the putative amino acid translation (1275 a.a.)of PGP-A cDNA (which corresponds to SEQ ID NO:6).

[0020]FIGS. 8A to 8B represent the partial cDNA sequence (3512 bp) ofthe 3′ end of the related but different PGP-O clone from the H.contortus cDNA library (which corresponds to SEQ ID NO:7).

[0021]FIG. 9 represents the partial cDNA sequence (2681 bp) of 3′ end ofthe related but different PGP-B clone from the H. contortus cDNA library(which corresponds to SEQ ID NO:8).

[0022]FIG. 10 shows the autoradiographs of the Southern blots of genomicDNA extracted from eggs of ivermectin sensitive and resistant strains ofH. contortus (MKIS AND MKIR) after digestion with PvuII, electrophoresisand probed with the [³²P]-432 bp H. contortus Pgp probe.

[0023]FIG. 11 shows the restriction length polymorphism of PCR productsfrom the DNA of individual male adult worms from ivermectin susceptible(lanes 1-9) or resistant (lanes 11-20) H. contortus strains, generatedwith P-glycoprotein primers PGP2S and PGPAS followed by digestion withDdeI and separation on non-denaturing polyacrylamide gelelectrophoresis. The arrows point to the three digestion fragments thatare associated with resistance.

[0024]FIGS. 12A and 12B represent the nucleic acid sequences comprisingsense primer PGP2S (FIG. 12A, which corresponds to SEQ ID NO:9) andantisense primer PGPAS (FIG. 12B, which corresponds to SEQ ID NO:10)which are constructed from the nematode P-glycoprotein homolog cDNAclone PGP-O-3′ (53 bp intron region) and are used to generate PCRproducts that are diagnostic for macrocyclic lactone endectocideresistance.

[0025]FIGS. 13A and 13B illustrate the efficacy of moxidectin (MOX)against H. contortus susceptible (FIG. 13A) or moxidectin-resistant(FIG. 13B) strains in jirds.

[0026]FIGS. 14A and 14B illustrate the efficacy of ivermectin (IVM)against H. contortus susceptible (FIG. 14A) and moxidectin-resistant(FIG. 14B) strains in jirds.

[0027]FIGS. 15A and 15B illustrate the efficacy of verapamil (VRP) withor without ivermectin (IVM; LD₅₀) against H. contortus susceptible (FIG.15A) or moxidectin-resistant (FIG. 15B) strains in jirds.

[0028]FIGS. 16A and 16B illustrate the efficacy of the combination ofmoxidectin (MOX; FIG. 16A) or ivermectin (IVM; FIG. 16B) with verapamil(VRP) against H. contortus moxidectin-resistant strain in jirds.

[0029]FIGS. 17A, 17B and 17C illustrate, respectively, the HinfIdigestion of P-glycoprotein PCR fragments from the DNA of individualworms of susceptible, ivermectin-resistant and moxidectin-resistant H.contortus, using primers PGP2S and PGPAS, followed by digestion andseparation on non-denaturing polyacrylamide gel electrophoresis. Thearrows on the right side of FIGS. 17B and 17C point to the digestionfragments that are associated with resistance while the arrows on theleft side point to the position and size of the standard markers.

[0030]FIGS. 18A, 18B and 18C illustrate, respectively, the AluIdigestion of P-glycoprotein PCR fragments from the DNA of individualworms of susceptible, ivermectin-resistant and moxidectin-resistant H.contortus, using primers PGP2S and PGPAS, followed by digestion andseparation on non-denaturing polyacrylamide gel electrophoresis. Thearrows on the right side of FIGS. 18B and 18C point to the digestionfragments that are associated with resistance while the arrows on theleft side point to the position and size of the standard markers.

DETAILED DESCRIPTION OF THE INVENTION

[0031] In accordance with the present invention, there are providednovel purified and isolated nucleic acid molecules encoding newP-glycoprotein homologs or the fragments thereof which regulate themacrocyclic lactone resistance. These nucleic acids find use as probesin innovative methods for the early diagnosis of a developing resistanceto the endectocides. In the past, there have been no DNA or RNA basedmethods of detection of macrocyclic lactone resistance available. Now,the present invention uniquely provides the genetic basis of theresistance and the diagnosis of resistance using nucleic acid probes.The early detection under the guidance of this invention allows formaintaining adequate control of parasites and maintaining the usefulnessof the macrocyclic lactone compounds. Additionally, the mechanism ofresistance to macrocyclic lactones can be used in development of screensfor identifying new antiparasitic agents.

[0032] The novel methods of the present invention which are useful fordetecting the resistance to macrocyclic lactone compounds in nematodesor arthropod pests utilize the new nucleic acid probes described herein.A variety of techniques well-known to those versed in the art can beemployed for the analysis. Desirably, the method detects changes ingenomic DNA or mRNA to provide a viable means for diagnosis ofmacrocyclic lactone resistance.

[0033] These methods include, for example, Polymerase Chain Reaction(PCR), hybridization in a Southern blot, Dot blot or Northern blotanalysis or the use of an antibody to a sequence of peptidescorresponding to the translation of the nucleotide sequences between thenovel primers of the invention of an individual pest or mixture of thepests such as worms, using primers or probes, for example, correspondingto the portion of the cDNA sequence of PGP-O between the sequencesidentified as PGP2S and PGPAS (see FIGS. 12A and 12B). Alternativeprimers or probes within this region which can be utilized in themethods of the invention include, but are not limited to, allcombinations of PCR primers or probes within this region or that ofother PGP homolog sequences such as PGP-A, PGP-B, PGP-O and the like.Basically, the coding region of the P-glycoprotein homolog genescorresponding to the cDNA sequences identified as PGP-A, PGP-A-3′,PGP-B, PGP-B-3′, PGP-O, PGP-O-3′ and the like is detected by PCR,Southern blot, Dot blot, Northern blot, Restriction Fragment LengthPolymorphism (RFLP) and other standard means of analysis. Surprisingly,it has been found that the digestion pattern from the PCR fragment, theblot data or the antibody-antigen reaction are associated withsusceptible or resistant traits which are diagnostic for the developmentof macrocyclic lactone resistance.

[0034] Polymerase Chain Reaction (PCR) can be employed for the detectionof resistance to the macrocyclic lactone compounds by synthesizing anucleic acid product which can be probed in conjunction with theSouthern blot analysis or initially digested with a restriction enzymefor RFLP analysis as described herein. The primers are used to initiatea PCR reaction using the nucleic acids extracted from the pest specimen.They are used to synthesize a P-glycoprotein sequence or sequences. ThePCR products can then be cut with restriction enzymes and the digestedsequences run on an electrophoresis gel. Examples of suitablerestriction enzymes that can be employed in the digestion of the PCRproducts include, but are not limited to, AluI, DdeI, HinfI, RsaI andthe like. The pattern of bands observed on a Southern blot or a Northernblot indicates which P-glycoprotein alleles are present in a pestspecimen such as the worm or group of worms. Some of the alleles can beassociated with macrocyclic lactone sensitivity and others withresistance to macrocyclic lactones. The PCR products, followed byrestriction enzyme digests, provide viable means for the detection ofresistance. The process of cutting the PCR products or the nucleic acidssuch as DNA for the RFLP analysis greatly increases the sensitivity andspecificity of the diagnosis.

[0035] Reverse Transcriptase—Polymerase Chain Reaction analysis (RT-PCR)can similarly be employed for the detection of resistance to themacrocyclic lactone compounds in nematode or arthropod pests. Typically,RNA from a nematode or arthropod specimen is extracted and reversetranscriptase followed by PCR, as described herein, is used to detectresistance.

[0036] By way of illustration, the nucleic acids, typically DNA for thePCR procedure or mRNA for RT-PCR, are extracted from the pest specimen,a pest known to be resistant to the macrocyclic lactone compounds and apest known to be susceptible to the macrocyclic lactones. The nucleicacids derived from the resistant and the susceptible pests are used as apoint of reference. The DNA, or cDNA produced by mRNA by ReverseTranscriptase, is denatured and the primers of the invention are addedto form a mixture. The three mixtures are subjected to many cycles ofPCR, usually digested by a restriction enzyme and subjected to gelelectrophoresis. Subsequently, the pattern and the intensity of thebands from the specimen to that of the reference nucleic acids, i.e.,DNA or cDNA, of the resistant and susceptible extracts are compared todetect the resistant population. Optionally, hybridization by a probe ofthe invention or use of a dye such as ethidium bromide to assist invisualizing the bands is included in the process.

[0037] Novel probes are used in the diagnosis of macrocyclic lactoneresistance by detecting susceptibility or resistance to the macrocycliclactones in the PCR assay. The primers which are used in the PCR assayare constructed, for example, from the nucleic acid sequences for theparasite P-glycoprotein homolog cDNA clones. Examples of suitable PCRprimers that can be employed in the PCR analysis are the primers PGP2Sand PGPAS used in the sense and antisense directions, respectively,which are constructed from PGP-O-3′ or PGP-O (see FIGS. 12A and 12B).The primers can also be prepared from the full or partial sequences ofother P-glycoprotein nucleic acids such as PGP-A, PGP-A-3′, PGP-B-3′,PGP-O, etc. and the complementary strands thereof which contain theregion found to be diagnostic of macrocyclic lactone resistance.Alternative useful sequences can be obtained by conventional means suchas hybridization techniques under standard or stringent conditions.

[0038] Southern blot, Dot blot or Northern blot may be prepared with thenucleic acid molecules from the nematode or the arthropod specimen and,using a probe comprising one of the nucleic acid molecule sequencesencoding for resistance or portion thereof, one can compare the level ofthe nucleic acids extracted from the specimen to the level of thenucleic acids from the probe, for example, by measuring or detecting thelevel of DNA or mRNA. Generally, three nucleic acid extracts are mappedto make the comparison: from the pest specimen, from a pest known to beresistant and from a pest known to be susceptible. In the case of theSouthern blot, the pattern of the bands is compared. With the Northernblot, either the pattern or the intensity of the bands is compared. Forthe Dot blot, the intensity of the spots is compared.

[0039] Another technique involves conducting a Restriction FragmentLength Polymorphism analysis (RFLP) by extracting the nucleic acids froma nematode or arthropod specimen, digesting the nucleic acid with arestriction enzyme, using a probe comprising one of the nucleic acidmolecule sequences encoding for resistance or portion thereof andcomparing the digestion pattern to that of the digestion pattern ofnematodes or arthropods known to be from populations either resistant orsensitive to the macrocyclic lactone endectocides. When DNA is cut withthe restriction enzyme, run on a gel and probed under the RFLPtechnique, the probe hybridizes with the similar sequences, but theirlength will vary depending upon where the restriction sites for thatenzyme occurs. By repeating the analysis with DNA from individual worms,slightly different patterns are observed due to polymorphism. Specificpatterns are diagnostic for the resistance gene. PvuII is an example ofa preferred restriction enzyme that can be employed for RFLP analysis.Other conventional restriction enzymes known to those of ordinary skillin the art may be substituted in the method.

[0040] A further example of a process useful in the present inventionfor detecting resistance concerns making antibodies which employ thenovel PGP protein homologs. For instance, an antibody may be prepared toa sequence of the peptide corresponding to the amino acid translation ofthe nucleic acids or the fragment thereof encoding the P-glycoproteinhomologs which regulate resistance. Then, a specimen of the nematode orthe arthropod pest, or the extract thereof, is prepared for reactionwith the above antibody. The specimen or the extract is reacted with theantibody under suitable conditions that allow antibody-antigen bindingto occur and, thereafter, the presence of the antibody-antigen bindingis detected by conventional methods.

[0041] The above-described methods for the detection of resistance tothe macrocyclic lactone compounds can optionally use a P-glycoproteinspecific ligand or dye. Usually, the level of the P-glycoprotein in thespecimen can more easily be observed using the ligand or dye andcompared to the levels obtained in known macrocyclic lacrone resistantand susceptible populations of nematodes or arthropods. The ligand ordye is usually radiolabelled so that it can be readily detected.Examples of suitable ligands useful in this method include, but are notlimited to, prazosin, azidoprazosin, iodoaryl-azidoprazosin and thelike. A variety of conventional dyes may be employed such as, forinstance, rhodamine 123, ethidium bromide and others.

[0042] For purposes of this invention, the nucleic acid molecule may beDNA, cDNA or RNA. However, in the most preferred embodiment of thisinvention, the nucleic acid probe is a cDNA molecule. Many of theforegoing methods illustrate extracted nucleic acids from Haemonchuscontortus. It is contemplated that the present invention embraces theuse of recombinant nucleic acids encoding for resistance orsusceptibility to the macrolides as well as isolated nucleic acids fromother worm strains or pest species.

[0043] The plasmids containing cDNA derived from Haemonchus contortusare deposited in connection with the present patent application andmaintained pursuant to the Budapest Treaty in the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.The cDNA sequences described herein are contained within plasmids(PBLUESCRIPT® II, commercially available from Stratagene Inc., La Jolla,Calif.) transformed into XLI-blue Escherichia coli bacterial strains.The plasmids identified as PGP-B-3′, PGP-O-3′ and PGP-A-5′ have beendeposited in the ATCC on Jan. 29, 1997 and have been assigned ATCCDesignation Numbers 98307, 98309 and 98310, respectively. The plasmidPGP-A-3′ has been deposited in the ATCC on Feb. 26, 1997 and has beenassigned ATCC Designation Number 98336. It should be appreciated thatother plasmids, which may be readily constructed using site-directedmutagenesis and the techniques described herein, are also encompassedwithin the scope of the present invention.

[0044] The present invention further relates to the unique reversal ofresistance in parasites to the macrocyclic lactone compounds byadministering or applying multidrug resistance reversing agents. Thisreversal of an existing resistance problem permits regainingsatisfactory parasite control. The nematode or arthropod parasites orpests of this invention refer to crop insects, crop or mammaliannematodes, arthropod ectoparasites and endoparasites of mammalsincluding acarids and the like.

[0045] Desirably, the multidrug resistance reversing agent is a calciumchannel blocker such as verapamil, nifedipine and the like; a calmodulinantagonist such as trifluoperazine, prochlorperazine and the like; avinca alkaloid analog such as vindoline, thaliblastine and the like; asteroidal agent such as progesterone and the like; a hormonal agent suchas tamoxifen, estradiol and the like; an immunosuppressive agent such ascyclosporin A, SDZ-PSC 833 and the like, an antibiotic such aserythromycin, cefoperazone, ceftriaxone, tetracycline and the like;miscellaneous compounds such as dipyridamole, quinidine, reserpine,amiodarone, etc.; and other multidrug resistance reversing agents knownto those versed in the art.

[0046] To increase the efficacy of the parasiticidal macrolides, thecompounds of the invention are administered to mammals orally,parenterally, topically (local activity) or transdermally (systemicactivity) depending upon the bioavailability of the selected medicinalby the desired route of administration. Parenteral administration of themedicinals encompasses any means other than orally, such as, forexample, intravenously, intramuscularly, subcutaneously,intratracheally, intraruminally, etc. It is apparent that theMDR-reversing agents are administered in connection with theadministration of the macrocyclic lactone compound encounteringresistance in the nematodes or the arthropod ectoparasites orendoparasites of mammals. However, the administration of theMDR-reversing agents may be made either before or during concurrentadministration of the macrocyclic lactones. If the MDR-reversing agentwill be given before the endectocide, medical or veterinary personnelcan readily determine by appropriate blood levels how far in advance theMDR-reversing agent may be given for increasing the macrolide'sefficacy. Typically, the MDR-reversing agent will be administered within24 hours of the start of endectocidal therapy and, preferably, within 4hours before or concomitantly with administering the macrocycliclactone.

[0047] In terms of dosage, the suitable amount of the MDR-reversingagent which is effective to increase the efficacy of the macrocycliclactone compound against resistant nematodes or resistant arthropodectoparasites or endoparasites will typically vary within a wide rangeof amounts at a variety of concentrations. The particular MDR-reversingagent selected for use with the specific endectocide will clearly affectthe useful dose of the MDR-reversing agent. It is contemplated thatselection of appropriate dosages of each MDR-reversing agent and themacrocyclic lactone compound to achieve the pesticidal enhancingeffective amount can be easily titrated by routine testing known tothose having ordinary skill in the medical and veterinary arts.

[0048] For use in parasiticidal treatment, the macrocyclic lactonecompounds may be administered orally in a unit dosage form such as acapsule, a bolus or a tablet. The capsules and boluses comprise theactive ingredient admixed with a conventional carrier vehicle such asstarch, talc, magnesium stearate or dicalcium phosphate. The dry, solidunit dosage form are prepared by intimately and uniformly mixing theactive ingredient with suitable finely divided diluents, fillers,disintegrating agents and/or binders such as starch, lactose, talc,magnesium stearate, vegetable gums and the like. Such unit dosageformulations may be widely varied with respect to their total weight andcontent of the active agent depending upon factors such as the type andthe weight of the mammal to be treated and the type and severity of theinfection or infestation. Generally, the amount of the macrocycliccompound given in oral administration is about 0.001 mg to about 10 mgper kg of body weight and preferably, about 1 mg to about 5 mg per kg ofbody weight. However, the amount will vary depending upon the extent ofthe resistance already developed in the parasite.

[0049] For animals, the macrocyclic lactone compound and many of theMDR-reversing agents can also be administered via an animal feedstuff byintimately dispersing the active ingredient in the feed or using as atop dressing or in the form of pellets which may then be added to thefinished feed or optionally fed separately. Suitable compositionsinclude feed premixes or supplements in which the active compound ispresent in relatively large amounts, wherein said feed premixes orsupplements are suitable for direct feeding to the animal or foraddition to the feed either directly or after an intermediate dilutionor blending step.

[0050] Typical carriers or diluents suitable for such compositionsinclude distillers' dried grains, corn meal, citrus meal, fermentationresidues, ground oyster shells, wheat products, molasses, corn cob meal,edible bean mill feed, soya grits, crushed limestone and the like. Theactive compounds are intimately dispersed throughout the carrier bymethods such as grinding, stirring, milling or tumbling. Compositionscontaining about 0.005% to about 2.0%, by weight, of the active compoundare particularly suitable as feed premixes.

[0051] Feed supplements, which are fed directly to the animal, containabout 0.0002% to 0.3%, by weight, of the active compounds. Suchsupplements are added to the animal feed in an amount to give thefinished feed the concentration of active compound desired for thetreatment or control of the resistant parasitic disease. Although thedesired concentration of the active compound will vary depending upon avariety of factors such as the particular compound employed or theseverity of the affliction, the macrocyclic compounds of this inventionare usually fed at concentrations of about 0.00001% to about 0.02% inthe feed.

[0052] Alternatively, the compounds of the present invention may beadministered to the afflicted mammals parenterally, in which event theactive ingredient is dissolved, dispersed or suspended in a sterile,isotonic, nontoxic liquid carrier vehicle. The active material isadmixed with the nontoxic pharmaceutically acceptable vehicle,preferably a vegetable oil such as peanut oil, cotton seed oil or thelike. Other parenteral vehicles such as propylene glycol, glycerol andthe like may also be used for parenteral formulations.

[0053] In the parenteral formulations, the active macrolides aretypically dissolved or suspended in the formulation in sufficient amountto provide from about 0.005% to about 5.0%, by weight, of the activecompound in said formulation.

[0054] Conveniently, the macrolides may also be administered to theafflicted mammals by the topical or transdermal route to achieve eitherlocal or systemic effect. When used on animals, the compounds may beapplied as a liquid drench. The animal drench is normally a solution,suspension or dispersion of the active compound, usually in water,together with a suspending agent such as bentonite and a wetting agentor similar excipient. Generally, the drenches also contain anantifoaming agent. Drench formulations typically contain about 0.001% toabout 0.5%, by weight, of the active macrocyclic compound. Preferreddrench formulations contain about 0.01% to about 0.1%, by weight.

[0055] Additionally, the macrocyclic compounds may be administered byapplying as a gel, lotion, solution, cream or ointment to human skin orpouring on animal skin or hide via a solution. The topical ortransdermal formulations comprise the active ingredient in combinationwith conventional inactive excipients and carriers. The cream, forexample, may use liquid petrolatum, white petrolatum, propylene glycol,stearyl alcohol, cetyl alcohol, sodium lauryl sulfate, sodium phosphatebuffer, polysorbates, parabens, emulsifying wax,polyoxyethylene-polyoxypropylene block copolymers, purified water andthe like. Ointments, for example, may employ petrolatum, mineral oil,mineral wax, glycerin and the like. Topical solutions may provide theactive ingredient compounded with propylene glycol, parabens,hydroxypropyl cellulose, preservatives. Pour-on formulations mayconstitute the active ingredient dissolved in a suitable inert solvent,such as dimethylsulfoxide, propylene glycol, butoxyethoxyethanol and thelike. A particularly useful pour-on formulation comprises the activeingredient dissolved or dispersed in an aromatic solvent, PPG-2 myristylether propionate, polybutene, an antimicrobial agent, an antioxidant anda nontoxic pharmaceutically acceptable mineral or vegetable oil.

[0056] To increase the efficacy of the macrolides as pesticidal agents,the multidrug resistance reversing agents are applied to crops, cropseeds or the soil or water in which crops or seeds are growing or to begrown in a pesticidal enhancing effective amount. The MDR-reversingagents may be applied either before or concurrently with the applicationof the macrocyclic lactone. Typically, the MDR-reversing agent will beapplied within 4 hours before or, preferably, concomitantly with theapplication of the macrocyclic lactone.

[0057] In terms of application rates, the suitable amount of theMDR-reversing agent which is effective to increase the efficacy of themacrocyclic lactone compound against resistant crop pests will typicallyvary within a wide range of amounts at a variety of concentrations andrates. The particular MDR-reversing agent selected for use with the croppesticide will clearly affect the application rate of the MDR-reversingagent. It is contemplated that choice of appropriate amounts,concentrations, spray rates and the like of each MDR-reversing agent andthe macrocyclic lactone compound to achieve the pesticidal enhancingeffective amount can be easily determined by routine procedures known tothose having ordinary skill in the agricultural art.

[0058] As insecticidal, nematocidal or acaricidal agents useful forprotecting crop seeds or growing or harvested crops from the pest'sattack, the compounds of the present invention may be formulated intodry compacted granules, flowable compositions, wettable powders, dusts,dust concentrates, microemulsions and the like, all of which lendthemselves to soil, water or foliage application and provide therequisite plant protection. Such compositions include the compounds ofthe invention admixed with agronomically acceptable solid or liquidcarriers.

[0059] In the agricultural composition, the active compounds areintimately mixed or ground together with the excipients and carriers insufficient amounts to typically provide from about 3% to about 20% byweight of the macrocyclic lactone compound in said composition.

[0060] The compositions of this invention are useful in combattingagricultural pests that inflict damage upon crops while they are growingor while in storage. The compounds are applied using known techniquessuch as sprays, dusts, emulsions, wettable powders, flowables and thelike to the growing or stored crops to provide protection againstinfestation by agricultural pests.

[0061] Unexpectedly, it is found that the mechanism of resistance to themacrocyclic lactone compounds is due to overexpression of novelP-glycoprotein homologs which causes an efflux of anthelmintic from theparasite. The present invention illustrates the involvement of the Pgphomolog genes in IVM resistance in H. contortus. The overexpression ofPgp-protein in IVM resistant strains of H. contortus is shown to beregulated by both rearrangement of genomic DNA encoding the PGP homologsand by gene transcription.

[0062]H. contortus in jirds (Meriones unguiculatus) has been used forthe evaluation of anthelmintic efficacy and has been shown to correlatewell with studies of this parasite in sheep (Conder et al., J.Parasitol. 78: 492-497, 1992). Employing the jird model, the presentinvention determines that multidrug reversing agents can unexpectedly beused to increase the efficacy of macrocyclic lactones against resistantparasites. As a representative example, the multidrug resistance (MDR)reversing agent verapamil (VRP) is shown to uniquely enhance the actionof moxidectin and ivermectin against moxidectin susceptible andresistant H. contortus.

[0063] Parasites such as H. contortus contain Pgp homolog genes whichare expressed in different stages of the parasite life cycle. Thisinvention finds that the level of expression of P-glycoprotein issurprisingly elevated in different strains that are resistant tomacrocyclic lactones such as ivermectin compared with the levels in thesusceptible strains from which the resistant strains are derived. Thehigher level of Pgp expression, in ivermectin resistant strains, isassociated with an alteration at the genomic level.

[0064] P-glycoproteins can act as molecular pumps to efflux hydrophobicxenobiotics from cells. An elevation in the level of the P-glycoproteinsis the basis of multidrug resistance in cancer cells and also appears tobe involved in some forms of drug resistance in some protozoa. Anelevated level of Pgp has not so far been described as the mechanism ofdrug resistance in nematode parasites. This is the first evidence thatshows that ivermectin resistance can be due to an elevation inP-glycoproteins. Ivermectin resistance is becoming a common problem innematode parasites of animals and potentially in arthropod parasites.Its continued use against arthropods is likely to lead to the selectionof similar resistance to that in nematodes.

[0065] Evidence exists that ivermectin shares a common action with otheravermectins, such as doramectin, milbemycins (Arena et al., 1995) andmoxidectin. It can be predicted that the development of resistanceagainst other macrocyclic lactone compounds will involve hyperexpressionof P-glycoprotein leading to elevated rates of drug efflux.

[0066] This work is significant because it allows the sensitivedetection of ivermectin resistance and resistance to other macrocycliclactone compounds in nematodes and arthropods using DNA and cDNA probesbased on the demonstrated differences found in the PvuII digests of DNAfrom resistant and susceptible organisms. It also permits the predictionof the degree of resistance from the level of P-glycoprotein expressionbased on either Pgp mRNA or Pgp protein levels. This understanding ofthe mechanism of resistance to the macrolides allows active analogs tobe synthesized which will remain effective in the presence of anmdr-based mechanism of resistance to other macrocyclic lactones. Morespecifically, chemicals which act on the mode of action receptor, theglutamate-gated chloride channel (Arena et al., 1995), but which are notefficiently effluxed by the P-glycoprotein pump, i.e., are poorsubstrates for Pgp efflux, can be selected to overcome resistance. Thiswill lead to improvements in parasite controls, especially in theprevention and treatment of ivermectin resistance and cross-resistanceto other avermectins, milbemycins and the LL-F28249 compounds.

[0067] This invention provides new evidence that in resistance tomacrocyclic lactone endectocides, such as ivermectin, in nematode andarthropod parasites of animals, expression of P-glycoprotein is elevatedcompared with the level of expression in the parental susceptiblestrains of the parasite. It further shows that the higher level ofexpression is associated with differences, at the genomic level, ofP-glycoprotein genes. For example, using Southern blot analysis ofpooled DNA and by PCR (Polymerase Chain Reaction) analysis of individualworms, differences are determined in the genomic DNA for Pgp inHaemonchus contortus resistant to ivermectin compared with thesusceptible parental strain, and the allele diversity for Pgp inresistant worms appears to be markedly reduced compared with theparental susceptible strain. Novel nucleic acid probes which candifferentiate between susceptible and resistant parasites are now foundand deemed to be useful in the early detection of the development ofresistance to macrocyclic lactone compounds.

[0068] For the first time, this invention demonstrates that macrolactoneresistance can be overcome by using a MDR-reversing agent. For example,verapamil, a well-known, relatively weak MDR-reversing agent,significantly increases the efficacy of moxidectin against moxidectinresistant H. contortus. The moxidectin resistant worms show sideresistance to ivermectin and the ivermectin resistance is also overcomewith the use of a mild MDR-reversing agent.

[0069] The following examples demonstrate certain aspects of the presentinvention. However, it is to be understood that these examples are forillustration only and do not purport to be wholly definitive as toconditions and scope of this invention. It should be appreciated thatwhen typical reaction conditions (e.g., temperature, reaction times,etc.) have been given, the conditions which are both above and below thespecified ranges can also be used, though generally less conveniently.The examples are conducted at room temperature (about 23° C. to about28° C.) and at atmospheric pressure. All parts and percents referred toherein are on a weight basis and all temperatures are expressed indegrees centigrade unless otherwise specified.

[0070] A further understanding of the invention may be obtained from thefollowing non-limiting examples.

EXAMPLE 1 PCR Synthesis and Cloning of a 432 by DNA for a P-GlycoproteinHomolog From a cDNA Library of H. contortus

[0071] Based on the highly conserved ATP binding domains of C. elegansPgp, a pair of degenerate PCR primers is designed. The sense primer is5′-ACNGTNGCNYTNGTNGG-3′ (which corresponds to SEQ ID NO:11) and theantisense primer is 5′-GCNSWNGTNGCYTCRTC-3′ (which corresponds to SEQ IDNO:12). PCR is carried out for 40 cycles at a denaturing temperature of94° C. for 1 minute, an annealing temperature of 37° C. for 1 minute,and an extension temperature of 72° C. for 3 minutes using an H.contortus cDNA library (Geary et al., Mol. Biochem. Parasitol., 50:295-306, 1992) as template. A 432 bp product is purified by agarose gelelectrophoresis and the purified product is used as template for asecond round of PCR amplification with the same primers. An enriched 432bp product is subsequently cloned into TA vector (Invitrogen) accordingto standard protocols. Plasmids with inserts are transformed intoEscherichia coli and then plated on Ampicillin LB plates containing achromogenic substrate, X-GAL®(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, commerciallyavailable from Gibco BRL, Bethesda, Md.) (Sambrook et al., MolecularCloning. A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1989). Ten clones are identified as ATP binding domain sequencesof P-glycoprotein.

EXAMPLE 2 Screening of the H. contortus cDNA Library

[0072] The 432 bp fragment is excised by EcoRI, labelled by randompriming with [³²P]d-CTP and used as a probe to screen the cDNA library(Sambrook et al., 1989). Approximately one million clones are screenedand nine putative clones are identified. The positive clones aredigested with PvuII and three of them containing inserts in thepredicted size are subsequently sequenced.

EXAMPLE 3 Parasite Strains

[0073] Two pairs of ivermectin susceptible and resistant strains of H.contortus are used. The first pair is an ivermectin resistant strain(MFR) developed at the Merck Research Laboratories, Rahway, N.J. (Rohreret al., J. Parasitol. 80: 493-497, 1994) and the ivermectin susceptibleparent strain (MFS) from which the resistant strain is selected overseventeen generations of ivermectin selection. The second pair is anivermectin resistant strain (ACR) developed at American CyanamidCompany, Princeton, N.J. and the ivermectin susceptible parent strain(ACS) from which the resistant strain is selected over fourteengenerations of ivermectin selection. Strain MFR is reported to be 10×resistant at the ED₉₅ compared with MFS, and ACR, after twelvegenerations of selection, is found to be 6.3× resistant at the ED₉₅compared with ACS.

EXAMPLE 4 RNA Extraction and Northern Hybridization

[0074] Adult worms from ivermectin susceptible and resistant H.contortus are collected from the abomasum of sheep (Lubega and Prichard,Biochem. Pharmacol. 41: 93-101, 1991). Eggs from each strain arecollected and isolated from faeces of sheep (Weston et al., J.Parasitol. 14: 159-164, 1984) which have been previously worm free andinoculated with one of the four H. contortus strains. Total RNA isextracted from tissues of the ivermectin susceptible and resistantstrains, respectively, using TRIzoL® Reagent (Gibco BRL LifeTechnologies, Inc., Gaithersburg, Md., company protocol). Total RNA isrun on denaturing formaldehyde agarose gel electrophoresis andtransferred to H-bond nylon membranes. The membranes are prehybridizedat 65° C. in 10% dextran disulfate, 1% SDS (sodium dodecylsulfate),11.0M NaCl over 4 hours. The ³²P-labelled 432 bp H. contortus Pgpfragment and an actin probe consisting of the 1.25 kb PstI fragment frompBAl (Degen et al., J. Biol. Chem. 258: 12153, 1983) are mixed andincubated overnight with the membranes at 65° C. in the samehybridization buffer. The membranes are washed with 2× SSC (1:2 mixtureof trisodium citrate and sodium chloride), 0.1% SDS at 65° C. for 30minutes and 0.5× SSC at 35° C. for 1 hour and then autoradiographed.Image analyses of gel autoradiographs are made for quantitativedetermination of mRNA expression, using the IMAGE program (O'Neil etal., Appl. Theor. Electrophor., 1: 163-167, 1989). Actin DNA probes froma mouse source, labelled and hybridized with the same blot, using thesame method as above, is used as an internal control for mRNA loading.Results are shown in FIG. 3 (S=unselected strains; R=IVM selectedstrains; MKI=strains developed at Merck Research Laboratories, Rahway,N.J.; ACI=strains developed at American Cyanamid Company, Princeton,N.J.).

EXAMPLE 5 Southern Blots

[0075] Genomic DNA from both ivermectin susceptible and resistantstrains is extracted (Sambrook et al., 1989). Four restriction enzymesEcoRI, ClaI, PvuII and PstI are used to digest the genomic DNA followingthe suppliers' directions. Each reaction is carried out with bothivermectin resistant and susceptible strains. After an overnightrestriction enzyme digestion, samples are run on 1% agarose gels andthen blotted onto H-bond membranes (Sambrook et al., 1989). Themembranes with DNA are exposed under UV light to fix the DNA to themembranes. The membranes are prehybridized at 65° C. in buffer (10%sulfur dextran, 1% SDS and 1M NaCl) for at least 4 hours. The 432 bpfragment, labelled with [³²P], is added as a probe and hybridized withthe genomic DNA in the prehybridization buffer, overnight. The membranesare subsequently washed twice with 2×SCC for 10 minutes, twice with1×SCC for 15 minutes and then autoradiographed.

Results PCR Amplification

[0076] Two rounds of PCR amplification generate a 432 bp product(FIG. 1) which is highly homologous to the conserved ATP binding domainof P-glycoprotein (FIG. 2A). The putative amino acid sequence (FIG. 2B)shows that this fragment is highly homologous to P-glycoprotein ormultiple drug resistant proteins from C. elegans, mouse and otherspecies. These data indicate that the 432 bp fragment represents the ATPbinding sequence of an H. contortus Pgp homolog.

Expression of P-Glycoprotein mRNA in Ivermectin Resistant andSusceptible Strains of H. contortus

[0077] A single animal species may have different Pgp which may vary insize. Northern hybridization, with the 432 bp Pgp H. contortus homologPCR product, shows that the molecular size of the mRNA for the H.contortus Pgp is about 4 kb. However, it is found that the mRNA levelsof Pgp in ivermectin resistant and susceptible strains of H. contortusare different. For illustration, results of a number of representativeNorthern blots on H. contortus egg RNA are shown in FIG. 3.

[0078] The RNA is also probed with an actin probe to allow correctionfor different amounts of RNA loaded onto the gels. The intensity of thePgp mRNA band varies with the strain of parasite. After correction forthe intensity of the actin band, it is found that the amount of the 4 kbmRNA band recognized by the 432 bp Pgp probe is much higher in bothivermectin resistant strains compared with their respective ivermectinsusceptible precursor strains. The increase varies from 250% to 670%after standardization for actin mRNA expression in drug resistant andsusceptible strains (Table 1). Similar results are also obtained incomparisons of Pgp expression using RNA extracted from adult H.contortus.

[0079] Table 1 shows the relative intensity of mRNA for P-glycoproteinand actin in ivermectin susceptible and resistant Haemonchus contortusstrains. RNA is extracted from eggs from the respective Merck (MKI) andAmerican Cyanamid (ACI) paired strains. Each susceptible and resistantpair are processed at the same time. The RNA is separated on an agarosegel and probed with both H. contortus 432 bp Pgp and the actin pBA1radiolabelled probes. The relative intensity of each band is determined,after gel autoradiography, by gel densitometry. The intensity of eachPgp band is corrected for intensity of its corresponding actin band inorder to adjust for different amounts of RNA having been loaded onto thegels. All comparisons are made by pairs (resistant (R) versuscorresponding susceptible (S)). TABLE 1 Strains comparison Corrected R/Sratio MKIS/MKIR 6.77 MKIS/MKIR 6.08 MKIS/MKIR 2.57 ACIS/ACIR 4.19

Sequencing of P-Glycoprotein Homologs From the H. contortus cDNA Library

[0080] Longer clones (4.2 kb, 3.5 kb and 2.7 kb) identified using the432 bp probe, which are shown to be homologous to P-glycoprotein, arefully or partially sequenced. FIGS. 4A to 4B show the full cDNA sequencefor the PGP-A clone (4175 bp) which has high homology to knownP-glycoprotein genes such as the Xenopus putative multidrug resistanceprotein (Xemdr) and the C. elegans cepgpA gene for P-glycoprotein A.FIGS. 5 and 6 show the partial sequence, in the sense direction (FIG. 5;PGP-A-5′) and the antisense direction (FIG. 6; PGP-A-3′) of the cDNAfragment which is also highly homologous to P-glycoprotein. FIGS. 7-9illustrate, respectively, the putative amino acid translation of PGP-AcDNA, the partial cDNA sequence of the 3′ end of the PGP-O clone (3.5kb), antisense direction, and the partial cDNA sequence of 3′ end of thePGP-B clone (2.7 kb), antisense direction.

Genomic DNA Differences Between Ivermectin Resistant and SusceptibleStrains of H. contortus and Determination of a Nucleic Acid Probe forthe Detection of Macrolactone Susceptibility or Resistance

[0081] Genomic DNA hybridizations show that at least two bands arerecognized by the 432 bp probe in ClaI and PstI digestion maps of bothivermectin susceptible and resistant strains. The EcoRI digestion mapsshow three strongly hybridizing bands and one light band for bothsusceptible and resistant strains. However, the PvuII digestion patternsare clearly different between the ivermectin resistant and susceptiblestrains (FIG. 10).

[0082] PCR products are generated using pairs of primers which arespecific to parasite Pgp genes. In one example, the reverse primer isspecific for a region 53 base pairs in length present in one of the Pgpclones (PGP-O). The forward primer anneals to a region common tomultiple Pgp clones. Genomic DNA extracted from individual male H.contortus adults from IVM-sensitive (24 worms) and IVM-resistant (29worms) populations (MKIS and MKIR) is used as template for amplificationby PCR. The Pgp PCR products, approximately 900 bp in length, aredigested with the restriction enzyme DdeI and the digestion products areseparated by non-denaturing polyacrylamide gel electrophoresis (FIG. 11;see also FIGS. 17A-18C illustrating diagnostic restriction patterns forresistance after selection with either ivermectin or moxidectin, usingdifferent worm strains and different restriction enzymes). The digestionpattern for the worms from the susceptible population is variable, whilethat for the worms from the resistant population is more homogeneous. Anidentical digestion pattern of three bands (arrows) is found in 28 ofthe 29 worms from the resistant population (FIG. 11, lanes 11-18 and 20,for example), whereas only 4 or 5 worms from the susceptible populationhave this pattern (FIG. 11, lanes 6 and 9, for example). Examples of theprobes are shown in FIGS. 12A and 12B.

[0083] These results are repeated several times. The PCR data and theSouthern blot data clearly indicate that selection for macrocycliclactone endectocide resistance causes a reduction in the geneticdiversity of the Pgp alleles and that the differences in Pgp at the DNAlevel can be detected by specific probes techniques such as PCR(Polymerase Chain Reaction), Southern blot analysis and RFLP(Restriction Fragment Length Polymorphism).

Additional Methods EXAMPLE 6 Establishing the LD₅₀ for Moxidectin andIvermectin Against Moxidectin Susceptible and Resistant H. contortus inthe Jird

[0084] Jirds, which are fed on a standard commercial ration to which0.02% hydrocortisone has been added 5 days prior to infection, areinoculated with 1000 exsheathed H. contortus. On day 10 afterinoculation, the jirds are treated with either water or various doses ofmoxidectin or ivermectin orally. Each treatment group contains 6 jirds.The parasite strains and anthelmintic dose rates are shown in Table 2.The results of these dose titrations are shown in FIGS. 13A-14B. Probitanalyses are used to estimate LD₅₀ levels for each anthelmintic againsteach strain. The estimated LD₅₀ of moxidectin against the susceptibleand moxidectin resistant strains are 0.010 and 0.017 mg/kg,respectively, and for ivermectin the estimate LD₅₀ levels are 0.024 and0.046 mg/kg, respectively.

[0085] The results indicate that (i) moxidectin is more potent thanivermectin against both the susceptible and moxidectin resistantstrains, and (ii) moxidectin resistant H. contortus are side-resistantto ivermectin. TABLE 2 DOSE RATES (mg/kg) OF MOXIDECTIN (MOX) ORIVERMECTIN (IVM) AGAINST H. contortus MOX-RESISTANT AND SUSCEPTIBLESTRAINS IN JIRDS (n = 6) COMPOUND SUSCEPTIBLE RESISTANT (mg/kg) (PF14)(MOF14) CONTROL — — MOX 0.0125 0.0125 MOX 0.025 0.025 MOX 0.05 0.05 MOX0.1 0.1 IVM 0.025 0.025 IVM 0.1 0.1 IVM 0.4 0.4 IVM 1.6 1.6

EXAMPLE 7 Determination of the Toxicity and the Efficacy of VerapamilAlone and in Combination With Ivermectin

[0086] This experiment is performed to determine the toxicity ofverapamil, a weak MDR-reversing agent, alone and in combination withivermectin, the efficacy of verapamil alone against H. contortus and theeffect of verapamil at 20 mg/kg on the efficacy of ivermectin againstsusceptible and moxidectin resistant worms. Dose rates of verapamilbetween 20 and 80 mg/kg are used alone or in combination with ivermectinat 0.024 and 0.046 mg/kg in jirds infected with susceptible ormoxidectin resistant H. contortus. Verapamil is given concomitantly withivermectin by the oral route. The results are shown in Table 3. TABLE 3DEMONSTRATION OF TOXICITY AND EFFICACY OF VERAPAMIL (VRP) WITH ORWITHOUT IVERMECTIN AGAINST H. contortus MOXIDECTIN RESISTANT ORSUSCEPTIBLE STRAINS IN JIRDS (n = # per group) COMPOUND # SUSCEPTIBLE #RESISTANT (mg/kg) (PF14) (MOF14) CONTROL 5 5 VRP 20 5 5 VRP 40 3 3 VRP60 3 3 VRP 80 3 3 IVM** 5 5 IVM/VRP 20 5 5 IVM/VRP 40 5 5 IVM/VRP 60 5 5IVM/VRP 80 5 5

[0087] As no deaths or other signs of toxicity are observed at averapamil dose rate of 20 mg/kg, in the absence or presence ofivermectin, this dose rate is used for subsequent resistance reversingexperiments. Verapamil alone is found to have no significant effect onworm counts at any of the dose rates used.

[0088] The toxicity of verapamil is summarized in Table 4. TABLE 4TOXICITY OF VERAPAMIL TO JIRDS VRP (mg/kg) DEATHS-VRP ALONE DEATHS-VRP +IVM* 20  0/10 0/10 40 0/5 2/10 60 1/6 1/10 80 3/7 2/10

[0089] Because of the toxicity of verapamil at dose rates of 40 mg/kgand above, only the effects of verapamil at 20 mg/kg on the efficacy ofivermectin against susceptible and moxidectin resistant worms areconsidered. These results, shown in FIGS. 15A and 15B, are summarized inTable 5. Verapamil at 20 mg/kg significantly enhances the efficacy ofivermectin against the moxidectin resistant worms. TABLE 5 EFFECT OFVERAPAMIL (20 mg/kg) ON THE EFFICACY (%) OF IVERMECTIN STRAINSUSCEPTIBLE MOX-RESISTANT (PF14) (MOF14) TREATMENT (mg/kg) % EFFICACY* %EFFICACY* CONTROL 0 0 VRP 20 17 (n.s.) −53 (n.s.) IVM^(#) 54 (A) 79 (A)IVM^(#)/VRP 20 92 (A) 96 (B)

EXAMPLE 8 The Effects of Verapamil on the Efficacy of Moxidectin andIvermectin Against Susceptible and Moxidectin Resistant H. contortus

[0090] This experiment is performed on jirds to determine the effects ofverapamil at 20 mg/kg on the efficacy of moxidectin and ivermectinagainst susceptible and moxidectin resistant H. contortus. Alltreatments have 7 jirds/group. The dose rates of moxidectin andivermectin are selected to give approximately 50% efficacy in theabsence of verapamil. Verapamil at 20 mg/kg significantly increases theefficacy of moxidectin against the resistant worms. The increaseobserved when verapamil is coadministered with ivermectin is notsignificant in this experiment as the efficacy obtained with ivermectinalone is already relatively high. The results are shown in Table 6 (seegraphic representation of results in FIGS. 16A and 16B). TABLE 6 EFFECTOF VERAPAMIL ON THE EFFICACY (%) OF MOXIDECTIN AND IVERMECTIN AGAINSTTHE MOXIDECTIN-RESISTANT STRAIN OF H. contortus (MOF14) EFFICACY (%)WORM COUNTS SIGNIFICANCE TREATMENT (MEAN ± S.E.) AT P < 0.05 PLACEBO 46± 7 —  A# VRP* 80 ± 9 −73 A MOX (0.017 mg/kg) 14 ± 3 70 B MOX (0.017mg/kg) +  2 ± 1 96 C VRP* IVM (0.028 mg/kg)  9 ± 1 80 B IVM (0.028mg/kg) +  3 ± 1 93 B VRP*

[0091] This experiment confirms that the weak MDR-reversing agentverapamil overcomes resistance in nematodes to the macrolactones. Theseresults are fully consistent with the above molecular evidence thatmacrolactone resistance is associated with the overexpression ofP-glycoprotein homolog due to a change in P-glycoprotein DNA inresistant parasites. More potent MDR-reversing agents, such ascyclosporin A, SDZ-PSC 833 or other potent reversing agents can, at lowdose rates, markedly increase the efficacy of macrocyclic lactoneendectocides against resistant parasites.

[0092] In the foregoing, there has been provided a detailed descriptionof particular embodiments of the present invention for the purpose ofillustration and not limitation. It is to be understood that all othermodifications, ramifications and equivalents obvious to those havingskill in the art based on this disclosure are intended to be includedwithin the scope of the invention as claimed.

1 12 432 base pairs nucleic acid double linear cDNA 1 ACGGTGGCGTTTGTTGGGCA GTCTGGTTGT GGAAAAAGCA CTGTGAAGGC GTTGTTGGAC 60 GGTTTTACAATCAAAACAAG GGCGTGATTA CGGACGCCGA AAACATCAGA AACATGAACA 120 TACGCAATCTTCGTGAGCAA GTGTGTATTG TAAGCCAGGA ACCAACGCTG TTCGACTGTA 180 CCATCATGGAAAACATCTGT TACGGTCTCG ATCGACCCCA AGCTCCTACG AACAGGTTGT 240 TGCTGCAGCAAAATCGGTCG AGTCGAAATG GCGAACATTC ACAATTTTGT GCTGGGACTA 300 CCAGAGGGTTACGATACGCG TGTTGGTGAG AAAGGCACTC AGCTGTCAGG CGGACAGAAG 360 AAACGAATAGCCATAGCCAG AGCGCTGATT CGAGATCCGC CTATACTTCT GCTGGATGAG 420 GCTACGACGG CC432 144 amino acids amino acid single linear protein 2 Thr Val Ala PheVal Gly Gln Ser Gly Cys Gly Lys Ser Thr Val Lys 1 5 10 15 Ala Leu LeuGlu Arg Phe Tyr Asn Gln Asn Lys Gly Val Ile Thr Asp 20 25 30 Ala Glu AsnIle Arg Asn Met Asn Ile Arg Asn Leu Arg Glu Gln Val 35 40 45 Cys Ile ValSer Gln Glu Pro Thr Leu Phe Asp Cys Thr Ile Met Glu 50 55 60 Asn Ile CysTyr Gly Leu Asp Asp Pro Lys Leu Leu Arg Thr Gly Cys 65 70 75 80 Cys CysSer Lys Ile Gly Arg Val Glu Met Ala Asn Ile His Asn Phe 85 90 95 Val LeuGly Leu Pro Glu Gly Tyr Asp Thr Arg Val Gly Glu Lys Gly 100 105 110 ThrGln Leu Ser Gly Gly Gln Lys Lys Arg Ile Ala Ile Ala Arg Ala 115 120 125Leu Ile Arg Asp Pro Pro Ile Leu Leu Leu Asp Glu Ala Thr Thr Ala 130 135140 4175 base pairs nucleic acid double linear cDNA 3 GGTTTAATTACCCAAGTTTG AGAGATCGTT CTCAAGCTGG TAAAATGTTC GAAAAAGGCC 60 AAGATGATGAACGTATACCA TTACTCGGTT CATCCAAGAA AAGTTCAATC GGCGAAGTCA 120 GTAAAAAAGAAGAACCGCCT ACAATAACAA ACCGTGGAAT TCTCTCCTTA GCCACTACAT 180 TGGATTATGTGCTTCTTGCG GCTGGTACGC TGGCGCCGTG TGTTCATGGC GCTGGATTCT 240 CAGTACTCGGTATTGTACTC GGTGGTATGA CGACAGTCTT TCTCAGAGCT CAGAACTCAG 300 AATTCGTTCTGGGCACTGTT AGTCGGGATC CTGAAGGGCT ACCAGCTCTT ACTAAGGAAG 360 AATTTGACACACTAGTACGT AGGTATTGCT TATACTACCT TGGATTAGGC TTTGCTATGT 420 TTGCAACATCTTATATACAG ATTGTGTGTT GGGAGACGTT CGCCGAACGA ATTACCCATA 480 AATTACGAAAAATTTATCTA AAAGCCATAC TTCGGCAGCA GATCTCATGG TTTGACATTC 540 AACAAACAGGAAATCTCACA GCTCGTCTAA CCGATGATCT CGAACGTGTT CGTGAAGGAC 600 TTGGTGATAAACTGTCGCTT TTTATACAAA TGGTGTCTGC TTTTGTGGCT GGTTTCTGTG 660 TAGGATTCGCGTATAGCTGG TCAATGACGC TCGTGATGAT GGTCGTGGCG CCGTTTATAG 720 TTATTTCTGCTAATTGGATG TCAAAAATCG TTGCTACTAG GACCCAAGTT GAACAGGAAA 780 CCTACGCTGTTGCCGGTGCT ATAGCGGAGG AGACTTTCTC ATCGATACGA ACCGTACACT 840 CGATATGTGGCCATAAAAGA GAGCTAACAA GATTTGAGGC AGCGTTGGAG AAAGGACGTC 900 AGACAGGCCTTGTCAAATAT TTCTATATGG GTGTTGGTGT GGGATTTGGT CAGATGTGTA 960 CCTATGTGTCCTACGCCTTG GCTTTTTGGT ATGGCAGTGT ACTGATCATC AACGACCCTG 1020 CATTGGATCGTGGCCGAATT TTCACAGTCT TTTTTGCTGT GATGTCCGGC TCAGCAGCTC 1080 TCGGCACATGTCTGCCACAT CTTAACACCA TATCCATCGC TCGAGGAGCG GTACGAAGTG 1140 TACTGTCAGTGATTAATAGT CGTCCAAAAA TCGATCCCTA TTCGTTAGAT GGCATTGTGC 1200 TCAACAATATGAGAGGATCT ATCCGCTTCA AGAACGTGCA TTTCTCCTAT CCTTCCCGAA 1260 GAACATTGCAGATATTGAAA GGTGTGTCAC TGCAAGTGTC GGCTGGCCAA AAAATTGCTT 1320 TGGTGGGTTCAAGCGGTTGT GGAAAGTCAA CGAACGTCAA TTTATTATTG AGATTTTATG 1380 ATCCGACAAGGGGAAAGGTA ACCATAGATG ATATTGATGT GTGTGATCTC AACGTGCAAA 1440 AACTTCGTGAACAAATCGGT GTTGTTAGTC AGGAACCAGT GCTTTTCGAT GGCACACTAT 1500 TCGAAAATATCAAGATGGGT TATGAACAGG CCACAATGGA GGAGGTCCAA GAAGCGTGCC 1560 GTGTGGCGAATGCTGCCGAC TTCACCAAAC GACTTCCAGA AGGTTACGGC ACCCGAGTTG 1620 GTGAACGTGGTGTGCAGTTA AGTGGCGGAC AAAAGCAGCG AATTGCCATA GCTCGTGCGA 1680 TCATCAAGAACCCTCGCATA CTGCTGCTCG ATGAAGCCAC CAGTGCTCTA GACACAGAAG 1740 CGGAATCAATCGTGCAAGAG GCTCTGGAGA AGGCTCAAAA AGGGAGAACA ACCGTCATTG 1800 TAGCGCATCGTCTGTCTACT ATCAGAAACG TGGATCAGAT TTTCGTTTTC AAGAACGGAA 1860 CGATCGTTGAGCAGGGCACT CATGCCGAGT TGATGAACAA ACGTGGAGTA TTCTTTGAAA 1920 TGACTCAAGCACAAGTCCTC CGACAAGAGA AGGAAGAGGA AGTTTTAGAT AGCGATGCGG 1980 AATCCGATGTCGTGTCACCG GATATTGCAT TACCCCATCT TAGTTCACTT CGATCCCGTA 2040 AAGAATCCACAAGAAGTGCT ATCTCCGCGG TCCCCAGCGT TCGAAGTATG CAAATCGAAA 2100 TGGAGGACCTTCGTGCCAAA CCAACTCCAA TGTCGAAAAT TTTCTATTTT AACCGTGACA 2160 AATGGGGATATTTCATTTTG GGACTCATCG CCTGTATTAT TACTGGAACT GTTACACCGA 2220 CATTTGCAGTTTTATATGCG CAGATCATAC AGGTATACTC GGAACCTGTT GATCAAATGA 2280 AAGGCCATGTGCTGTTCTGG TGTGGAGCTT TCATCGTCAT TGGTCTCGTA CACGCTTTTG 2340 CGTTCTTTTTCTCGGCTATT TGTTTGGGAC GTTGCGGCGA AGCGTTAACG AAAAAATTAC 2400 GTTTCGAGGCGTTCAAGAAC CTTCTGCGAC AGAATGTGGG ATTCTACGAC GATATCCGAC 2460 ACGGTACCGGTAAACTCTGT ACGCGATTTG CTACAGATGC ACCCAATGTC CGATATGTGT 2520 TCACTCGACTTCCGGGTGTG CTTTCATCGG TGGTGACCAT AATTGGAGCT TTGGTTATTG 2580 GATTCATCTTCGGGTGGCAG CTGGCTTTGA TTCTTATGGT GATGGTACCG TTGATCATCG 2640 GTAGTGGATACTTCGAGATG CGCATGCAGT TTGGTAAGAA GATGCGTGAC ACAGAGCTTC 2700 TTGAAGAGGCTGGGAAAGTT GCCTCTCAAG CCGTGGAGAA CATTCGTACC GTGCATGCCC 2760 TGAATAGGCAAGAGCAGTTC CATTTCATGT ATTGCGAGTA TTTGAAGGAA CCCTATCGAG 2820 AAAATCTTTGCCAGGCGCAC ACCTACGGGG GTGTATTCGC GTTCTCACAA TCGTTGTTAT 2880 TCTTTATGTATGCTGTAGCA TTTTGGATTG GTGCAATCTT CGTGGACAAC CACAGCATGC 2940 AACCGATTGACGTTTACCGA GTATTTTTCG CGTTCATGTT TTGTGGACAA ATGGTCGGCA 3000 ACATTTCTTCTTTTATTCCT GACGTTGTGA AAGCTCGCCT GGCTGCATCG CTCCTTTTCT 3060 ACCTTATCGAACACCCATCA GAAATTGATA ATTTGTCCGA GGATGGTGTC ACGAAGAAAA 3120 TCTCTGGTCATATCTCGTTC CGCAATGTCT ATTTCAATTA TCCGACAAGA AGACAGATCA 3180 GAGTACTCCGTGGACTTAAC CTAGAGATAA ATCCTGGCAC GACGGTAGCG CTTGTTGGGC 3240 AGTCTGGTTGTGGAAAAAGC ACTGTGATGG CGTTGTTGGA ACGGTTTTAC AATCAAAACA 3300 AGGGCGTGATTACGGTGGAC GGCGAAAACA TCAGAAACAT GAACATACGC AATCTTCGTG 3360 AGCAAGTGTGTATTGTTAGC CAGGAACCAA CGCTGTTCGA CTGTACCATC ATGGAAAACA 3420 TCTGTTACGGTCTCGATGAC CCCAAGCCGT CCTACGAACA GGTTGTTGCT GCAGCAAAAA 3480 TGGCGAACATTCACAATTTT GTGCTGGGAC TACCAGAGGG TTACGATACG CGTGTTGGTG 3540 ARAAAGGCACTCAGCTGTCA GGCGGACAGA AGCAACGAAT AGCCATAGCC AGAGCGCTGA 3600 TTCGAGATCCGCCTATACTT CTGCTGGATG AGGCGACAAG CGCGCTGGAT ACCGAGAGTG 3660 AAAAGATCGTGCAAGACGCC CTAGAGGTTG CTCGCCAAGG TAGAACGTGC CTTGTAATTG 3720 CCCATCGCCTTTCTACAATT CAAGACAGTG ACGTCATAGT GATGATCCAG GAGGGGAAAG 3780 CTACAGACAGAGGCACTCAT GAACATTTAC TGATGAAGAA CGATCTATAC AAACGGCTAT 3840 GCGAAACACAACGACTCGTT GAATCACAAT GAGTTTTTAG TGCCAATCGA TAGTGATCGA 3900 TAAGCTATGGATTAGTCTTT AACACTTACT GATCATATGA CTCTATCTCG TGCTTTATTA 3960 TAATGTACATATGTAATGGT TTTGATCTTA CATATCTTGT AATTGGTCCT CACTATCATA 4020 ATGCCTTTAGTAGTATATTA ACAGTTTTAT TAATACAACT TAAGTAACAT ATTAACAATT 4080 TTATTAATATAACTTAAGTA AGATATTGAC AGTTTTATTA ATTTGGAGGA TTTATAATAA 4140 AACCTCGTGCCGCTCGTGCC GAAACGATAT CAAGC 4175 1810 base pairs nucleic acid doublelinear cDNA 4 GGTTTAATTA CCCAAGTTTG AGAGATCGTT CTCAAGCTGG TAAAATGTTCGAAAAAGGCC 60 AAGATGATGA ACGTATACCA TTACTCGGTT CATCCAAGAA AAGTTCAATCGGCGAAGTCA 120 GTAAAAAAGA AGAACCGCCT ACAATAACAA ACCGTGGAAT TCTCTCCTTAGCCACTACAT 180 TGGATTATGT GCTTCTTGCG GCTGGTACGC TGGCGCCGTG TGTTCATGGCGCTGGATTCT 240 CAGTACTCGG TATTGTACTC GGTGGTATGA CGACAGTCTT TCTCAGAGCTCAGAACTCAG 300 AATTCGTTCT GGGCACTGTT AGTCGGGATC CTGAAGGGCT ACCAGCTCTTACTAAGGAAG 360 AATTTGACAC ACTAGTACGT AGGTATTGCT TATACTACCT TGGATTAGGCTTTGCTATGT 420 TTGCAACATC TTATATACAG ATTGTGTGTT GGGAGACGTT CGCCGAACGAATTACCCATA 480 AATTACGAAA AATTTATCTA AAAGCCATAC TTCGGCAGCA GATCTCATGGTTTGACATTC 540 AACAAACAGG AAATCTCACA GCTCGTCTAA CCGATGATCT CGAACGTGTTCGTGAAGGAC 600 TTGGTGATAA ACTGTCGCTT TTTATACAAA TGGTGTCTGC TTTTGTGGCTGGTTTCTGTG 660 TAGGATTCGC GTATAGCTGG TCAATGACGC TCGTGATGAT GGTCGTGGCGCCGTTTATAG 720 TTATTTCTGC TAATTGGATG TCAAAAATCG TTGCTACTAG GACCCAAGTTGAACAGGAAA 780 CCTACGCTGT TGCCGGTGCT ATAGCGGAGG AGACTTTCTC ATCGATACGAACCGTACACT 840 CGATATGTGG CCATAAAAGA GAGCTAACAA GATTTGAGGC AGCGTTGGAGAAAGGACGTC 900 AGACAGGCCT TGTCAAATAT TTCTATATGG GTGTTGGTGT GGGATTTGGTCAGATGTGTA 960 CCTATGTGTC CTACGCCTTG GCTTTTTGGT ATGGCAGTGT ACTGATCATCAACGACCCTG 1020 CATTGGATCG TGGCCGAATT TTCACAGTCT TTTTTGCTGT GATGTCCGGCTCAGCAGCTC 1080 TCGGCACATG TCTGCCACAT CTTAACACCA TATCCATCGC TCGAGGAGCGGTACGAAGTG 1140 TACTGTCAGT GATTAATAGT CGTCCAAAAA TCGATCCCTA TTCGTTAGATGGCATTGTGC 1200 TCAACAATAT GAGAGGATCT ATCCGCTTCA AGAACGTGCA TTTCTCCTATCCTTCCCGAA 1260 GAACATTGCA GATATTGAAA GGTGTGTCAC TGCAAGTGTC GGCTGGCCAAAAAATTGCTT 1320 TGGTGGGTTC AAGCGGTTGT GGAAAGTCAA CGAACGTCAA TTTATTATTGAGATTTTATG 1380 ATCCGACAAG GGGAAAGGTA ACCATAGATG ATATTGATGT GTGTGATCTCAACGTGCAAA 1440 AACTTCGTGA ACAAATCGGT GTTGTTAGTC AGGAACCAGT GCTTTTCGATGGCACACTAT 1500 TCGAAAATAT CAAGATGGGT TATGAACAGG CCACAATGGA GGAGGTCCAAGAAGCGTGCC 1560 GTGTGGCGAA TGCTGCCGAC TTCACCAAAC GACTTCCAGA AGGTTACGGCACCCGAGTTG 1620 GTGAACGTGG TGTGCAGTTA AGTGGCGGAC AAAAGCAGCG AATTGCCATAGCTCGTGCGA 1680 TCATCAAGAA CCCTCGCATA CTGCTGCTCG ATGAAGCCAC CAGTGCTCTAGACACAGAAG 1740 CGGAATCAAT CGTGCAAGAG GCTCTGGAGA AGGCTCAAAA AGGGAGAACAACCGTCATTG 1800 TAGCGCATCG 1810 2698 base pairs nucleic acid doublelinear cDNA 5 AGTGCTTTTC GATGGCACAC TATTCGAAAA TATCAAGATG GGTTATGAACAGGCCACAAT 60 GGAGGAGGTC CAAGAAGCGT GCCGTGTGGC GAATGCTGCC GACTTCACCAAACGACTTCC 120 AGAAGGTTAC GGCACCCGAG TTGGTGAACG TGGTGTGCAG TTAAGTGGCGGACAAAAGCA 180 GCGAATTGCC ATAGCTCGTG CGATCATCAA GAACCCTCGC ATACTGCTGCTCGATGAAGC 240 CACCAGTGCT CTAGACACAG AAGCGGAATC AATCGTGCAA GAGGCTCTGGAGAAGGCTCA 300 AAAAGGGAGA ACAACCGTCA TTGTAGCGCA TCGTCTGTCT ACTATCAGAAACGTGGATCA 360 GATTTTCGTT TTCAAGAACG GAACGATCGT TGAGCAGGGC ACTCATGCCGAGTTGATGAA 420 CAAACGTGGA GTATTCTTTG AAATGACTCA AGCACAAGTC CTCCGACAAGAGAAGGAAGA 480 GGAAGTTTTA GATAGCGATG CGGAATCCGA TGTCGTGTCA CCGGATATTGCATTACCCCA 540 TCTTAGTTCA CTTCGATCCC GTAAAGAATC CACAAGAAGT GCTATCTCCGCGGTCCCCAG 600 CGTTCGAAGT ATGCAAATCG AAATGGAGGA CCTTCGTGCC AAACCAACTCCAATGTCGAA 660 AATTTTCTAT TTTAACCGTG ACAAATGGGG ATATTTCATT TTGGGACTCATCGCCTGTAT 720 TATTACTGGA ACTGTTACAC CGACATTTGC AGTTTTATAT GCGCAGATCATACAGGTATA 780 CTCGGAACCT GTTGATCAAA TGAAAGGCCA TGTGCTGTTC TGGTGTGGAGCTTTCATCGT 840 CATTGGTCTC GTACACGCTT TTGCGTTCTT TTTCTCGGCT ATTTGTTTGGGACGTTGCGG 900 CGAAGCGTTA ACGAAAAAAT TACGTTTCGA GGCGTTCAAG AACCTTCTGCGACAGAATGT 960 GGGATTCTAC GACGATATCC GACACGGTAC CGGTAAACTC TGTACGCGATTTGCTACAGA 1020 TGCACCCAAT GTCCGATATG TGTTCACTCG ACTTCCGGGT GTGCTTTCATCGGTGGTGAC 1080 CATAATTGGA GCTTTGGTTA TTGGATTCAT CTTCGGGTGG CAGCTGGCTTTGATTCTTAT 1140 GGTGATGGTA CCGTTGATCA TCGGTAGTGG ATACTTCGAG ATGCGCATGCAGTTTGGTAA 1200 GAAGATGCGT GACACAGAGC TTCTTGAAGA GGCTGGGAAA GTTGCCTCTCAAGCCGTGGA 1260 GAACATTCGT ACCGTGCATG CCCTGAATAG GCAAGAGCAG TTCCATTTCATGTATTGCGA 1320 GTATTTGAAG GAACCCTATC GAGAAAATCT TTGCCAGGCG CACACCTACGGGGGTGTATT 1380 CGCGTTCTCA CAATCGTTGT TATTCTTTAT GTATGCTGTA GCATTTTGGATTGGTGCAAT 1440 CTTCGTGGAC AACCACAGCA TGCAACCGAT TGACGTTTAC CGAGTATTTTTCGCGTTCAT 1500 GTTTTGTGGA CAAATGGTCG GCAACATTTC TTCTTTTATT CCTGACGTTGTGAAAGCTCG 1560 CCTGGCTGCA TCGCTCCTTT TCTACCTTAT CGAACACCCA TCAGAAATTGATAATTTGTC 1620 CGAGGATGGT GTCACGAAGA AAATCTCTGG TCATATCTCG TTCCGCAATGTCTATTTCAA 1680 TTATCCGACA AGAAGACAGA TCAGAGTACT CCGTGGACTT AACCTAGAGATAAATCCTGG 1740 CACGACGGTA GCGCTTGTTG GGCAGTCTGG TTGTGGAAAA AGCACTGTGATGGCGTTGTT 1800 GGAACGGTTT TACAATCAAA ACAAGGGCGT GATTACGGTG GACGGCGAAAACATCAGAAA 1860 CATGAACATA CGCAATCTTC GTGAGCAAGT GTGTATTGTT AGCCAGGAACCAACGCTGTT 1920 CGACTGTACC ATCATGGAAA ACATCTGTTA CGGTCTCGAT GACCCCAAGCCGTCCTACGA 1980 ACAGGTTGTT GCTGCAGCAA AAATGGCGAA CATTCACAAT TTTGTGCTGGGACTACCAGA 2040 GGGTTACGAT ACGCGTGTTG GTGARAAAGG CACTCAGCTG TCAGGCGGACAGAAGCAMCG 2100 AATAGCCATA GCCAGAGCGC TGATTCGAGA TCCGCCTATA CTTCTGCTGGATGAGGCGAC 2160 AAGCGCGCTG GATACCGAGA GTGAAAAGAT CGTGCAAGAC GCCCTAGAGGTTGCTCGCCA 2220 AGGTAGAACG TGCCTTGTAA TTGCCCATCG CCTTTCTACA ATTCAAGACAGTGACGTCAT 2280 AGTGATGATC CAGGAGGGGA AAGCTACAGA CAGAGGCACT CATGAACATTTACTGATGAA 2340 GAACGATCTA TACAAACGGC TATGCGAAAC ACAACGACTC GTTGAATCACAATGAGTTTT 2400 TAGTGCCAAT CGATAGTGAT CGATAAGCTA TGGATTAGTC TTTAACACTTACTGATCATA 2460 TGACTCTATC TCGTGCTTTA TTATAATGTA CATATGTAAT GGTTTTGATCTTACATATCT 2520 TGTAATTGGT CCTCACTATC ATAATGCCTT TAGTAGTATA TTAACAGTTTTATTAATACA 2580 ACTTAAGTAA CATATTAACA ATTTTATTAA TATAACTTAA GTAAGATATTGACAGTTTTA 2640 TTAATTTGGA GGATTTATAA TAAAACCTCG TGCCGCTCGT GCCGAAACGATATCAAGC 2698 1275 amino acids amino acid single linear protein 6 MetPhe Glu Lys Gly Gln Asp Asp Glu Arg Ile Pro Leu Leu Gly Ser 1 5 10 15Ser Lys Lys Ser Ser Ile Gly Glu Val Ser Lys Lys Glu Glu Pro Pro 20 25 30Thr Ile Thr Asn Arg Gly Ile Leu Ser Leu Ala Thr Thr Leu Asp Tyr 35 40 45Val Leu Leu Ala Ala Gly Thr Leu Ala Pro Cys Val His Gly Ala Gly 50 55 60Phe Ser Val Leu Gly Ile Val Leu Gly Gly Met Thr Thr Val Phe Leu 65 70 7580 Arg Ala Gln Asn Ser Glu Phe Val Leu Gly Thr Val Ser Arg Asp Pro 85 9095 Glu Gly Leu Pro Ala Leu Thr Lys Glu Glu Phe Asp Thr Leu Val Arg 100105 110 Arg Tyr Cys Leu Tyr Tyr Leu Gly Leu Gly Phe Ala Met Phe Ala Thr115 120 125 Ser Tyr Ile Gln Ile Val Cys Trp Glu Thr Phe Ala Glu Arg IleThr 130 135 140 His Lys Leu Arg Lys Ile Tyr Leu Lys Ala Ile Leu Arg GlnGln Ile 145 150 155 160 Ser Trp Phe Asp Ile Gln Gln Thr Gly Asn Leu ThrAla Arg Leu Thr 165 170 175 Asp Asp Leu Glu Arg Val Arg Glu Gly Leu GlyAsp Lys Leu Ser Leu 180 185 190 Phe Ile Gln Met Val Ser Ala Phe Val AlaGly Phe Cys Val Gly Phe 195 200 205 Ala Tyr Ser Trp Ser Met Thr Leu ValMet Met Val Val Ala Pro Phe 210 215 220 Ile Val Ile Ser Ala Asn Trp MetSer Lys Ile Val Ala Thr Arg Thr 225 230 235 240 Gln Val Glu Gln Glu ThrTyr Ala Val Ala Gly Ala Ile Ala Glu Glu 245 250 255 Thr Phe Ser Ser IleArg Thr Val His Ser Ile Cys Gly His Lys Arg 260 265 270 Glu Leu Thr ArgPhe Glu Ala Ala Leu Glu Lys Gly Arg Gln Thr Gly 275 280 285 Leu Val LysTyr Phe Tyr Met Gly Val Gly Val Gly Phe Gly Gln Met 290 295 300 Cys ThrTyr Val Ser Tyr Ala Leu Ala Phe Trp Tyr Gly Ser Val Leu 305 310 315 320Ile Ile Asn Asp Pro Ala Leu Asp Arg Gly Arg Ile Phe Thr Val Phe 325 330335 Phe Ala Val Met Ser Gly Ser Ala Ala Leu Gly Thr Cys Leu Pro His 340345 350 Leu Asn Thr Ile Ser Ile Ala Arg Gly Ala Val Arg Ser Val Leu Ser355 360 365 Val Ile Asn Ser Arg Pro Lys Ile Asp Pro Tyr Ser Leu Asp GlyIle 370 375 380 Val Leu Asn Asn Met Arg Gly Ser Ile Arg Phe Lys Asn ValHis Phe 385 390 395 400 Ser Tyr Pro Ser Arg Arg Thr Leu Gln Ile Leu LysGly Val Ser Leu 405 410 415 Gln Val Ser Ala Gly Gln Lys Ile Ala Leu ValGly Ser Ser Gly Cys 420 425 430 Gly Lys Ser Thr Asn Val Asn Leu Leu LeuArg Phe Tyr Asp Pro Thr 435 440 445 Arg Gly Lys Val Thr Ile Asp Asp IleAsp Val Cys Asp Leu Asn Val 450 455 460 Gln Lys Leu Arg Glu Gln Ile GlyVal Val Ser Gln Glu Pro Val Leu 465 470 475 480 Phe Asp Gly Thr Leu PheGlu Asn Ile Lys Met Gly Tyr Glu Gln Ala 485 490 495 Thr Met Glu Glu ValGln Glu Ala Cys Arg Val Ala Asn Ala Ala Asp 500 505 510 Phe Thr Lys ArgLeu Pro Glu Gly Tyr Gly Thr Arg Val Gly Glu Arg 515 520 525 Gly Val GlnLeu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile Ala Arg 530 535 540 Ala IleIle Lys Asn Pro Arg Ile Leu Leu Leu Asp Glu Ala Thr Ser 545 550 555 560Ala Leu Asp Thr Glu Ala Glu Ser Ile Val Gln Glu Ala Leu Glu Lys 565 570575 Ala Gln Lys Gly Arg Thr Thr Val Ile Val Ala His Leu Arg Ser Thr 580585 590 Ile Arg Asn Val Asp Gln Ile Phe Val Phe Lys Asn Gly Thr Ile Val595 600 605 Glu Gln Gly Thr His Ala Glu Leu Met Asn Lys Arg Gly Val PhePhe 610 615 620 Glu Met Thr Gln Ala Gln Val Leu Arg Gln Glu Lys Glu GluGlu Val 625 630 635 640 Leu Asp Ser Asp Ala Glu Ser Asp Val Val Ser ProAsp Ile Ala Leu 645 650 655 Pro His Leu Ser Ser Leu Arg Ser Arg Lys GluSer Thr Arg Ser Ala 660 665 670 Ile Ser Ala Val Pro Ser Val Arg Ser MetGln Ile Glu Met Glu Asp 675 680 685 Leu Arg Ala Lys Pro Thr Pro Met SerLys Ile Phe Tyr Phe Asn Arg 690 695 700 Asp Lys Trp Gly Tyr Phe Ile LeuGly Leu Ile Ala Cys Ile Ile Thr 705 710 715 720 Gly Thr Val Thr Pro ThrPhe Ala Val Leu Tyr Ala Gln Ile Ile Gln 725 730 735 Val Tyr Ser Glu ProVal Asp Gln Met Lys Gly His Val Leu Phe Trp 740 745 750 Cys Gly Ala PheIle Val Ile Gly Leu Val His Ala Phe Ala Phe Phe 755 760 765 Phe Ser AlaIle Cys Leu Gly Arg Cys Gly Glu Ala Leu Thr Lys Lys 770 775 780 Leu ArgPhe Glu Ala Phe Lys Asn Leu Leu Arg Gln Asn Val Gly Phe 785 790 795 800Tyr Asp Asp Ile Arg His Gly Thr Gly Lys Leu Cys Thr Arg Phe Ala 805 810815 Thr Asp Ala Pro Asn Val Arg Tyr Val Phe Thr Arg Leu Pro Gly Val 820825 830 Leu Ser Ser Val Val Thr Ile Ile Gly Ala Leu Val Ile Gly Phe Ile835 840 845 Phe Gly Trp Gln Leu Ala Leu Ile Leu Met Val Met Val Pro LeuIle 850 855 860 Ile Gly Ser Gly Tyr Phe Glu Met Arg Met Gln Phe Gly LysLys Met 865 870 875 880 Arg Asp Thr Glu Leu Leu Glu Glu Ala Gly Lys ValAla Ser Gln Ala 885 890 895 Val Glu Asn Ile Arg Thr Val His Ala Leu AsnArg Gln Glu Gln Phe 900 905 910 His Phe Met Tyr Cys Glu Tyr Leu Lys GluPro Tyr Arg Glu Asn Leu 915 920 925 Cys Gln Ala His Thr Tyr Gly Gly ValPhe Ala Phe Ser Gln Ser Leu 930 935 940 Leu Phe Phe Met Tyr Ala Val AlaPhe Trp Ile Gly Ala Ile Phe Val 945 950 955 960 Asp Asn His Ser Met GlnPro Ile Asp Val Tyr Arg Val Phe Phe Ala 965 970 975 Phe Met Phe Cys GlyGln Met Val Gly Asn Ile Ser Ser Phe Ile Pro 980 985 990 Asp Val Val LysAla Arg Leu Ala Ala Ser Leu Leu Phe Tyr Leu Ile 995 1000 1005 Glu HisPro Ser Glu Ile Asp Asn Leu Ser Glu Asp Gly Val Thr Lys 1010 1015 1020Lys Ile Ser Gly His Ile Ser Phe Arg Asn Val Tyr Phe Asn Tyr Pro 10251030 1035 1040 Thr Arg Arg Gln Ile Arg Val Leu Arg Gly Leu Asn Leu GluIle Asn 1045 1050 1055 Pro Gly Thr Thr Val Ala Leu Val Gly Gln Ser GlyCys Gly Lys Ser 1060 1065 1070 Thr Val Met Ala Leu Leu Glu Arg Phe TyrAsn Gln Asn Lys Gly Val 1075 1080 1085 Ile Thr Val Asp Gly Glu Asn IleArg Asn Met Asn Ile Arg Asn Leu 1090 1095 1100 Arg Glu Gln Val Cys IleVal Ser Gln Glu Pro Thr Leu Phe Asp Cys 1105 1110 1115 1120 Thr Ile MetGlu Asn Ile Cys Tyr Gly Leu Asp Asp Pro Lys Pro Ser 1125 1130 1135 TyrGlu Gln Val Val Ala Ala Ala Lys Met Ala Asn Ile His Asn Phe 1140 11451150 Val Leu Gly Leu Pro Glu Gly Tyr Asp Thr Arg Val Gly Glu Lys Gly1155 1160 1165 Thr Gln Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile AlaArg Ala 1170 1175 1180 Leu Ile Arg Asp Pro Pro Ile Leu Leu Leu Asp GluAla Thr Ser Ala 1185 1190 1195 1200 Leu Asp Thr Glu Ser Glu Lys Ile ValGln Asp Ala Leu Glu Val Ala 1205 1210 1215 Arg Gln Gly Arg Thr Cys LeuVal Ile Ala His Arg Leu Ser Thr Ile 1220 1225 1230 Gln Asp Ser Asp ValIle Val Met Ile Gln Glu Gly Lys Ala Thr Asp 1235 1240 1245 Arg Gly ThrHis Glu His Leu Leu Met Lys Asn Asp Leu Tyr Lys Arg 1250 1255 1260 LeuCys Glu Thr Gln Arg Leu Val Glu Ser Gln 1265 1270 1275 3512 base pairsnucleic acid double linear cDNA 7 CCCGTTGTTG CCGGTGCTAT AGCGGAGGAGACTTTCTCAT CGATACGAAC CGTACACTCG 60 TTATGTGGCC ATAAAAGAGA GCTAACAAGGCAGCGTTGGA GAAAGGACGT CAGACAGGCC 120 TTGTCAAATA TTTCTATATG GGTGTTGGTGTGAGATTTGG TCAGATGTGT ACCTATGTGT 180 CCTACGCCTT GGCTTTTTGG TATGGCAGTGTACTGATCAT CAACGACCCT GCATTGGATC 240 GTGGCCGAAT TTTCACAGTC TTTTTGCTGTGATGTCCGGC TCAGCAGCTC TCGGCACATG 300 TCTGCCACAT CTTAACACCA TATCCATCGCTCGAGGAGCG GTACGAAGTG TACTGTCAGT 360 GATTAATAGT CGTCCAAAAA TCGATCCCTATTCGTTAGAT GGCATTGTGC TCAACAATAT 420 GAGAGGATCT ATTCGCTTCA AGAACGTGCATTTCTCCTAT CCTTCCCGAA GAACATTGCA 480 GATATTGAAA GGTGTGTCAC TGCAAGTGTCGGCTGGCCAA AAAATTGCTT TGGTGGGTTC 540 AAGCGGTTGT GGAAAGTCAA CGATCGTCAATTTATTATTG AGATTTTATG ATCCGACAAG 600 GGGAAAGGTA ACCATAGATG ATATTGATGTGTGTGATCTC AACGTGCAAA AACTTCGTGA 660 ACAAATCGGT GTTGTTAGTC AGGAACCAGTGCTTTTCGAT GGCACACTAT TCGAAAATAT 720 CAAGATGGGT TATGAACAGG CCACAATGGAGGAGGTCCAA GAAGCGTGCC GTGTGGCGAA 780 TGCTGCCGAC TTCATCAAAC GACTTCCAGAAGGTTACGGC ACCCGAGTTG GTGAACGTGG 840 TGTGCAGTTA AGTGGCGGAC AAAAGCAGCGAATTGCCATA GCTCGTGCGA TCATCAAGAA 900 CCCTCGCATA CTGCTGCTCG ATGAAGCCACCAGTGCTCTA GACACAGAAG CGGAATCAAT 960 CGTGCAAGAG GCTCTGGAGA AGGCTCAAAAAGGGAGAACA ACCGTCATTG TAGCGCATCG 1020 TCTGTCTACT ATCAGAAACG TGGATCAGATTTTCGTTTTC AAGAACGGAA CGATCGTTGA 1080 GCAGGGCACT CATGCCGAGT TGATGAACAAACGTGGAGTA TTCTTTGAAA TGACTCAAGC 1140 ACAAGTCCTC CGACAAGAGA AGGAAGAGGAAGTTTTAGAA AATACGGAAC CAGTAGCGAA 1200 GTGTCAAGAG GTATCCCTCC CTGCTCCTGATGTCACTATT TTGGCTCCCC ATGAGGAACA 1260 ACCCGAGCTA CCTAGCCCGC CGGGTCGGTTAGAAAATACA AAGCAACATG AGCATCTCTG 1320 AATGTCTTTG TCTGAGATAG CGATGCGGAATCCGATGTCG TGTCACCGGA TATTGCATTA 1380 CCCCATCTTA GTTCACTTCG ATCCCGTAAAGAATCCACAA GAAGTGCTAT CTCCGCGGTC 1440 CCCAGCGTTC GAAGTATGCA AATCGAAATGGAGGACCTTC GTGCCAAACC AACTCCAATG 1500 TCGAAAATTT TCTATTTTAA CCGTGACAAATGGGCATATT TCATTTTGGG ACTCATCGCC 1560 TGTATTATTA CTGGAACTGT TACACCGACATTTGCAGTTT TATATGCGCA GATCATACAG 1620 GTATACTCGG AACCTGTTGA TCAAATGAAAGGCCATGTGC TGTTCTGGTG TGGAGCTTTC 1680 ATCGTCATTG GTCTCGTACA CGCTTTTGCGTTCTTTTTCT CGGCTATTTG TTTGGGACGT 1740 TGCGGCGAAG CGTTAACGAA AAAATTACGTTTCGAGGCGT TCAAGAACCT TCTGCGACAG 1800 GATGTGGGAT TCTACGACGA TATCCGACACGGTACCGGTA AACTCTGTAC GCGATTTGCT 1860 ACAGATGCAC CCAATGTCCG ATATGTGTTCACTCGACTTC CGGGTGTGCT TTCATCGGTG 1920 GTGACCATAA TTGGAGCTTT GGTTATTGGATTCATCTTCG GGTGGCAGCT GGCTTTGATT 1980 CTTATGGTGA TGGTACCGTT GATCATCGGTAGTGGATACT TCGAGATGCG CATGCAGTTT 2040 GGTAAGGAGA TGCGTGACAC AGAGCTTCTTGAAGAGGCTG GGAAAGTTGC CTCTCAAGCC 2100 GTGGAGAACA TTCGTACCGT GCATGCCCTGAATAGGCAAG AGCAGTTCCA TTTCATGTAT 2160 TGCGAGTATT TGAAGGAACC CTATCGAGAAAATCTTTGCC AGGCGCACAC CTACGGGGGT 2220 GTATTCGCGT TCTCACAATC GTTGTTATTCTTTATGTATG CTGTAGCATT TTGGATTGGT 2280 GCAATCTTCG TGGACAACCA CAGCATGCAACCGATTGACG TTTACCGAGT ATTTTTCGCG 2340 TTCATGTTTT GTGGACAAAT GGTCGGCAACATTTCTTCTT TTATTCCTGA CGTTGTGAAA 2400 GCTCGCCTGG CTGCATCGCT CCTTTTCTACCTTATCGAAC ACCCATCAGA AATTGATAAT 2460 TTGTCCGAGG ATGGTGTCAC GAAGAAAATCTCTGGTCATA TCTCGTTCCG CAATGTCTAT 2520 TTCAATTATC CGACAAGAAG ACAGATCAGAGTACTCCGTG GACTTAACCT AGAGATAAAT 2580 CCTGGCACGA AGGTAGCGCT TGTTGGGCAGTCTGGTTGTG GAAAAAGCAC TGTGATGGCG 2640 TTGTTGGAAC GGTTTTACAA TCAAAACAAGGGCGTGATTA CGGTGGACGG CGAAAACATC 2700 AGAAACATGA ACATACGCAA TCTTCGTGAGCAAGTGTGTA TTGTAAGCCA GGAACCAACG 2760 CTGTTCGACT GTACCATCAT GGAAAACATCTGTTACGGTC TCGATGACCC CAAGCCGTCC 2820 TACGAACAGG TTGTTGCTGC AGCAAAAATGGCGAACATTC ACAATTTTGT GCTGGGACTA 2880 CCAGAGGGTT ACGATACGCG TGTTGGTGAGAAAGGCACTC AGCTGTCAGG CGGACAGAAG 2940 CAACGAATAG CCATAGCCAG AGCGCTGATTCGAGATCCGC CTATACTTCT GCTGGATGAG 3000 GCGACAAGCG CGCTGGATAC CGAGAGTGAAAAGATCGTGC AAGACGCCCT AGAGGTTGCT 3060 CGCCAAGGTA GAACGTGCCT TGTAATTGCCCATCGCCTTT CTACAATTCA AGACAGTGAC 3120 GTCATAGTGA TGATCCAGGA GGGGAAAGCTACAGACAGAG GCACTCATGA ACATTTACTG 3180 ATGAAGAACG ATCTATACAA ACGGCTATGCGAAACACAAC GACTCGTTGA ATCACAATGA 3240 GTTTTTAGTG CCAATCGATA GTGATCGATAAGCTATGGAT TAGTCTTTAA CACTTACTGA 3300 TCACAAATTT TATCTCGTGC TTTATTCTAATGTACATATG TAACGGTTTT GATCTTACAT 3360 ATCTTGTAAT TGGTCCTCAC TATCATAATGCCTTTAGTAG TACATTAACA GTTTTATTAA 3420 TACAACTTAA GTAACATATT AACAATTTTATTAATATAAC TTAAGTAAGA TATTGACAGT 3480 TTTATTAATT TGGAGGATTT ATAATAAAACTT 3512 2681 base pairs nucleic acid double linear cDNA 8 CCCGACTTCCGGAAGGTTAC GGCACCCGAG TAGGTGAACG TGGTGTACAA CTAAGTGGCG 60 GACAAAAGCAGCGCATCGCT ATTGCTCGCG CCATCATTAA AAACCCTCGT ATACTTCTGC 120 TTGACGAAGCCACCAGTGCT CTGGACACAG AGGCGGAATC AATTGTGCAA GAAGCTCTCG 180 AGAAAGCTCAAAAAGGACGA ACGACCGTCA TTGTAGCGCA TCGCCTATCT ACCATCAGAA 240 ATGTCGATCAAATTTTCGTC TTCAAGAATG AAACGATTGT TGAGCAGGGT ACACATGCAG 300 AGTTGATGAACAAACGAGGA GTGTTCTTTG AAATGACTCA AGCACAGGTC CTTCGACAAG 360 AAAAGGAAGAGGAGGTCTTA GAAAATACGG AACCAGTAGC GAAGTGTCAA GAGGCATCCT 420 TTCCTGCTCCTGATGTCACT ATTTTGACTC CCCATGACGA ACAACCCGAG CTACTTAGCC 480 CGCCGGATAGCGATGCGGAA TCCGACGTCA TGTCACCGGA TCTTGGCTTA CCCCATCTTA 540 GTTCACTTCGATCACGTAAA GAGTCCACAA GAAGTGCTAT TTCCGCAGTC CCCAGCGTTC 600 GGAGTATGCAGATCGAAATG GAGGACCTTC GTGCCAAACC GACTCCGATG TCGAAAATTT 660 TCTATTTCAACCGTGACAAA TGGGGATTTT TCATTTTGGG ACTCATCGCC TGTATTATAA 720 CTGGAACTGTTACACCGACA TTTGCAGTTT TATATGCGCA GATCATACAG GTATACTCGG 780 AACCTGTTGATCAAATGAAA GGCCATGTGC TGTTTTGGTG TGGAGCTTTC ATCGTCATTG 840 GTCTCGTACACGCATTTGCG TTCTTTTTCT CGGCCATTTG TCTGGGACGT TGCGGCGAAG 900 CTTTAACGAAGAAGTTACGT TTCGAGGCGT TCAAGAACCT TCTCCGACAA GATGTGGGAT 960 TCTACGACGATATCCGACAC GGTACCGGTA AACTCTGTAC GCGATTTGCT ACAGATGCAC 1020 CCAATGTTCGATATGTGTTC ACTCGACTTC CGGGTGTACT TTCATCGGTG GTGACCATAA 1080 TCGGAGCTTTGGTTATTGGA TTTATTTTCG GGTGGCAGCT GGCCTTGATT CTTATGGTCA 1140 TGGTACCGTTGATCATTGGC AGTGGATACT TCGAGATGCG CATGCAGTTT GGTAAAAAGA 1200 TGCGTGACACAGAGCTTCTT GAAGAGGCTG GGAAAGTTGC CTCACAAGCC GTAGAGAATA 1260 TTCGTACCGTACATGCCCTG AATCGGCAAG AGCAGTTCCA TTTCATGTAC TGCGAGTATT 1320 TGAAGGAACCCTATCGAGAG AATCTTTGCC AGGCGCACAC TTACGGGGGT GTATTCGCGT 1380 TTTCACAGTCGTTGTTATTC TTTATGTATG CTGTAGCATT TTGGATTGGT GCAATCTTCG 1440 TGGACAACCACAGCATGCAA CCGATTGATG TTTACCGAGT ATTTTTCGCG TTCATGTTTT 1500 GTGGACAAATGGTTGGCAAC ATTTCGTCCT TCATCCCTGA TGTTGTGAAA GCTCGCCTGG 1560 CTGCATCGCTCCTTTTCTAC CTCATCGAAC ACCCATCAGA AATTGATAAC TTGTCCGAGG 1620 ATGGTGTCAAGAAGAAAATC TCTGGTCACA TCTCGTTCCG CAATGTCTAT TTCAATTACC 1680 CGACGAGAAGGCAGATCAGA GTACTCCGTG GACTTAACCT AGAGATAAAT CCTGGCACGA 1740 CGGTAGCGCTTGTTGGACAA TCTGGTTGTG GAAAAAGCAC TGTGATGGCG TTGTTGGAAC 1800 GCTTTTACAATCAAAACAAG GGCGTGATTA CGGTTGACGG CGAAAACATC AGAAACATGA 1860 ACATACGCAATCTCCGTGAG CAAGTATGTA TAGTCAGCCA GGAACCAACA CTGTTCGACT 1920 GTACCATCATGGAAAACATC TGTTACGGAC TCGATGACCC CAAACCGTCC TACGAACAGG 1980 TTGTTGCGGCAGCAAAAATG GCGAACATCC ACAATTTTGT GCTGGGACTG CCAGAGGGTT 2040 ATGACACGCGTGTTGGCGAG AAAGGCACTC AGCTGTCAGG CGGACAAAAG CAAAGAATAG 2100 CCATAGCCCGAGCGCTGATC CGAGATCCGC CTATACTTCT GCTGGATGAG GCGACAAGCG 2160 CTCTGGACACGGAGAGTGAG AAGATCGTGC AAGACGCCCT AGAGGTTGCT CGCCAAGGTA 2220 GAACGTGCCTTGTAATTGCC CACCGCCTTT CTACAATTCA AGACAGTGAC GTCATAGTGA 2280 TGATCCAGGAGGGAAAAGCT ACAGACAGAG GCACTCATGA ACATTTACTG ATGAAGAACG 2340 ATCTATACAAACGGCTATGC GAAACACAAC GACTCGTTGA ATCACAATGA GTTTTTAGTG 2400 CCGATCGATAGTGATCGATA AGCTATGGAT TAGTCTTCAA CACTTACTGA TCATATGACT 2460 ATCTCGTGCTTTATTATAAT GTACATATGT AATGGTTTTG ATGTAAGTTA AGTTATAATT 2520 GGTCTTCACTATCATAATGC CTTTAGTAAT GCATTAACAC TTTTATAATA TAACTTGAAT 2580 AACATATTGACAGTTTTATT AATATAACTT AAATAAGATA TTGACAGTTT TATTAATTTG 2640 GAGAATTTATAATGAAACTT CTGGATTCCT GCAGCCCGGG G 2681 19 base pairs nucleic acidsingle linear cDNA 9 GAAATGACTC AAGCACAAG 19 18 base pairs nucleic acidsingle linear cDNA 10 AGACAAAGAC ATTCAGAG 18 17 base pairs nucleic acidsingle linear cDNA 11 ACNGTNGCNY TNGTNGG 17 17 base pairs nucleic acidsingle linear cDNA 12 GCNSWNGTNG CYTCRTC 17

We claim:
 1. A purified and isolated nucleic acid molecule or a fragmentthereof, extracted from a nematode or an arthropod pest, encoding aP-glycoprotein homolog which regulates resistance to a macrocycliclactone compound.
 2. The nucleic acid molecule or the fragment accordingto claim 1, wherein the nucleic acid molecule or the fragment isextracted from Haemonchus contortus.
 3. The nucleic acid molecule or thefragment according to claim 2, wherein the nucleic acid molecule or thefragment has a nucleotide sequence encoding PGP-A set forth in SEQ IDNO:3, PGP-A-3′ set forth in SEQ ID NO:5 (ATCC accession number 98336),PGP-B, PGP-B-3′ set forth in SEQ ID NO:8 (ATCC accession number 98307),PGP-O or PGP-O-3′ set forth in SEQ ID NO:7 (ATCC accession number98309); the complementary strands thereof or a nucleotide sequence whichhybridizes at about 65° C. in the presence of a dextran buffer over atleast about 4 hours to the nucleotide sequence encoding PGP-A, PGP-A-3′,PGP-B, PGP-B-3′, PGP-O or PGP-O-3′.
 4. A biologically functional plasmidor viral vector containing the nucleic acid molecule or the fragmentaccording to claim
 1. 5. A suitable host cell stably transformed ortransfected by a vector comprising the nucleic acid molecule or thefragment according to claim
 1. 6. A process for the production of apolypeptide product having part or all of the primary structuralconformation and the biological activity of a P-glycoprotein homologproduct, said process comprising: growing, under suitable nutrientconditions, procaryotic or eucaryotic host cells transformed ortransfected with a nucleic acid molecule or a fragment thereof,according to claim 1, in a manner allowing expression of saidpolypeptide product, and isolating the desired polypeptide product ofthe expression of said nucleic acid molecule or said fragment.
 7. AP-glycoprotein homolog product of the expression in a procaryotic oreucaryotic host cell of the nucleic acid molecule or the fragmentaccording to claim
 6. 8. A recombinant nucleic acid molecule or afragment thereof encoding a P-glycoprotein homolog which regulatesresistance to a macrocyclic lactone compound.
 9. The recombinant nucleicacid molecule or the fragment according to claim 8, wherein the nucleicacid molecule or the fragment has a nucleotide sequence encoding PGP-Aset forth in SEQ ID NO:3, PGP-A-3′ set forth in SEQ ID NO:5 (ATCCaccession number 98336), PGP-B, PGP-B-3′ set forth in SEQ ID NO:8 (ATCCaccession number 98307), PGP-O or PGP-O-3′ set forth in SEQ ID NO: 7(ATCC accession number 98309); the complementary strands thereof or anucleotide sequence which hybridizes at about 65° C. in the presence ofa dextran buffer over at least about 4 hours to the nucleotide sequenceencoding PGP-A, PGP-A-3′, PGP-B, PGP-B-3′, PGP-O or PGP-O-3′.
 10. Amethod for detecting the resistance to a macrocyclic lactone compound ina nematode or an arthropod pest which comprises comparing a nucleic acidmolecule extracted from a pest specimen to a nucleic acid moleculeencoding for resistance to the macrocyclic lactone compound and anucleic acid molecule encoding for susceptibility to the macrocycliclactone compound.
 11. The method according to claim 10, wherein thenucleic acid molecule extracted from the pest specimen is hybridizedwith a nucleic acid probe having a nucleotide sequence encoding PGP-Aset forth in SEQ ID NO:3, PGP-A-3′ set forth in SEQ ID NO:5 (ATCCaccession number 98336), PGP-B, PGP-B-3′ set forth in SEQ ID NO:8 (ATCCaccession number 98307), PGP-O or PGP-O-3′ set forth in SEQ ID NO:7(ATCC accession number 98309); the complementary strands thereof or anucleotide sequence which hybridizes at about 65° C. in the presence ofa dextran buffer over at least about 4 hours to the nucleotide sequenceencoding PGP-A, PGP-A-3′, PGP-B, PGP-B-3′, PGP-O or PGP-O-3′.
 12. Themethod according to claim 10, wherein one to three of the nucleic acidmolecules are mixed with a Polymerase Chain Reaction (PCR) or a ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) primer.
 13. The methodaccording to claim 12, in which the PCR or RT-PCR primer comprises anucleotide sequence between PGP2S and PGPAS in the sense and antisensedirections, respectively, set forth in FIGS. 12A and 12B or a nucleotidesequence encoding PGP-A set forth in SEQ ID NO:3, PGP-A-3′ set forth inSEQ ID NO:5 (ATCC accession number 98336), PGP-B, PGP-B-3′ set forth inSEQ ID NO:8 (ATCC accession number 98307), PGP-O or PGP-O-3′ set forthin SEQ ID NO:7 (ATCC accession number 98309); the complementary strandsthereof or a nucleotide sequence which hybridizes at about 65° C. in thepresence of a dextran buffer over at least about 4 hours to thenucleotide sequence encoding PGP-A, PGP-A-3′, PGP-B, PGP-B-3′, PGP-O orPGP-O-3′.
 14. A method for detecting the resistance to a macrocycliclactone compound in a nematode or an arthropod pest which comprisespreparing an antibody to a sequence of a peptide corresponding to theamino acid translation of a nucleic acid molecule or a fragment thereofencoding a P-glycoprotein homolog which regulates resistance to amacrocyclic lactone compound; preparing a specimen of the nematode orthe arthropod pest, or an extract thereof, for reaction with theantibody; reacting the specimen or the extract with the antibody undersuitable conditions that allow antibody-antigen binding to occur; andtesting for the presence of the antibody-antigen binding.
 15. A methodfor increasing the efficacy of a macrocyclic lactone compound against aresistant crop pest which comprises applying to the crop, to the cropseed or to the soil or water in which the crop or the seed is growing oris to be grown a pesticidal enhancing effective amount of a multidrugresistance reversing agent.
 16. A method for increasing the efficacy ofa macrocyclic lactone compound against a resistant nematode or aresistant arthropod ectoparasite or endoparasite of a mammal whichcomprises administering a pesticidal enhancing effective amount of amultidrug resistance reversing agent to the mammal in connection withthe administration of the macrocyclic lactone compound.
 17. The methodaccording to claim 15 or 16, wherein the multidrug resistance reversingagent is selected from the group consisting of a calcium channelblocker, a calmodulin antagonist, a vinca alkaloid analog, a steroidalagent, a hormonal agent, an immunosuppressive agent, an antibiotic anddipyridamole.
 18. The method according to claim 17, wherein themultidrug resistance reversing agent is verapamil, nifedipine,progesterone, tamoxifen, estradiol, cyclosporin A or SDZ-PSC
 833. 19.The method according to claim 15 or 16, wherein the macrocyclic lactonecompound is isolated from Streptomyces.
 20. The method according toclaim 15 or 16, wherein the macrocyclic lactone compound is selectedfrom the group consisting of LL-F28249α-λ, a 23-oxo derivative ofLL-F28249α-λ, a 23-imino derivative of LL-F28249α-λ, an avermectin, a22,23-dihydro derivative of avermectin and a milbemycin.
 21. The methodaccording to claim 15 or 16, wherein the macrocyclic lactone compound isLL-F28249α, 23-(o-methyloxime)-LL-F28249α,23-(O-methyloxime)-5-(phenoxyacetoxy)-LL-F28249α,23-(semicarbazone)-LL-F28249α, 23-(thiosemicarbazone)-LL-F28249α,ivermectin, abamectin, doramectin, eprinomectin, milbemycin A ormilbemycin D.
 22. The method according to claim 16, wherein the mammalis selected from the group consisting of a human, a sheep, a goat, acow, a deer, a horse, a swine, a dog and a cat.
 23. An improvedcomposition for controlling or combatting a crop pest which comprises apesticidally effective amount of a multidrug resistance reversing agent;a macrocyclic lactone compound selected from the group consisting ofLL-F28249α-λ, a 23-oxo derivative of LL-F28249α-λ, a 23-imino derivativeof LL-F28249α-λ, an avermectin, a 22,23-dihydro derivative of avermectinand a milbemycin; and an agronomically acceptable carrier.
 24. Animproved composition for controlling or treating helminth or arthropodendo- or ectoparasitic insect infestation or infection of a mammal whichcomprises an anthelmintically or an arthropod endo- orectoparasiticidally effective amount of a multidrug resistance reversingagent; a macrocyclic lactone compound selected from the group consistingof LL-F28249α-λ, a 23-oxo derivative of LL-F28249α-λ, a 23-iminoderivative of LL-F28249α-λ, an avermectin, a 22,23-dihydro derivative ofavermectin and a milbemycin; and a nontoxic pharmaceutically acceptablecarrier.
 25. The composition according to claim 23 or 24, wherein themultidrug resistance reversing agent is selected from the groupconsisting of a calcium channel blocker, a calmodulin antagonist, avinca alkaloid analog, a steroidal agent, a hormonal agent, animmunosuppressive agent, an antibiotic and dipyridamole.
 26. An improvedmethod for controlling or combatting a crop pest which comprisesapplying to the crop, to the crop seed or to the soil or water in whichthe crop or the seed is growing or is to be grown a pesticidallyeffective amount of the composition of claim
 23. 27. An improved methodfor controlling or treating helminth or arthropod endo- or ectoparasiticinsect infection or infestation of a mammal which comprisesadministering to the mammal to be treated an anthelmintically or anarthropod endo- or ectoparasiticidally effective amount of thecomposition of claim 24.